Inhibitors of cruzipain and other cysteine proteases

ABSTRACT

Compounds of general formula (I): 
                 
 
wherein R 1 , Y, (X) o , (W) n , (V) m , Z and U are as defined in the specification, are inhibitors of cruzipain and other cysteine protease inhibitors and are useful as therapeutic agents, for example in Chagas&#39; disease, or for validating therapeutic target compounds.

THIS INVENTION relates to compounds which are inhibitors of the proteasecruzipain, a gene product of the Trypanosoma cruzi parasite. Inparticular, the invention provides compounds that are useful for thetherapeutic treatment of Trypanosoma cruzi infection, to the use ofthese compounds, and to pharmaceutical compositions comprising them.Furthermore, this invention relates to compounds which are inhibitorsacross a broad range of cysteine proteases, to the use of thesecompounds, and to pharmaceutical compositions comprising them. Suchcompounds are useful for the therapeutic treatment of diseases in whichparticipation of a cysteine protease is implicated.

The trypanosomal family of parasites have a substantial worldwide impacton human and animal healthcare (McKerrow, J. H., et al, Ann. Rev.Microbiol. 47 821-853, 1993). One parasite of this family, Trypanosomacruzi, is the causative agent of Chagas' disease, which affects inexcess of twenty million people annually in Latin and South America, isthe leading cause of heart disease in these regions and results in morethan 45,000 deaths per annum (Centers for Disease Control and preventionwebsite). In addition, with the increase in migration of the infectedpopulation from rural to urban sites and movements from South andCentral America into North America, the infection is spreading via bloodtransfusions, and at birth. The present treatments of choice forTrypanosoma cruzi infection, nifurtimox and benznidazole (an NADHfumarate reductase inhibitor, Turrens, J F, et al, Mol BiochemParasitol., 82(1), 125-9, 1996) are at best moderately successful,achieving ˜60% cure during the acute phase of infection (see Docampo, R.Curr. Pharm. Design, 7, 1157-1164, 2001 for a general discussion) whilstnot being prescribed at all during the chronic phase wherecardiomyopathy associated heart failure often occurs (Kirchhoff, L. V.New Engl. J. Med., 329, 639-644, 1993). Additionally, these two drugshave serious adverse toxic effects, requiring close medical supervisionduring treatment, and have been shown to induce chromosomal damage inchagastic infants (Gorla, N. B. et al, Mutat. Res. 206, 217-220, 1988).Therefore, a strong medical need exists for new effective drugs for thechemotherapeutic treatment of Trypanosoma cruzi infection. Classically,the identification of enzymes found to be crucial for the establishmentor propagation of an infectious disease has been instrumental in thedevelopment of successful drugs such as antivirals (e.g. HIV aspartylprotease inhibitors) and anti-bacterials (e.g. β-lactam antibiotics).The search for a similar Achilles heel in parasitic infections hasexamined numerous enzymes (e.g. parasitic dihydrofolate reductase, seeChowdhury, S. F. et al, J. Med. Chem., 42(21), 4300-4312, 1999;trypanothione reductase, see Li, Z. et al, Bioorg. Med. Chem. Lett.,11(2), 251-254, 2001; parasitic glyceraldehydes-3-phosphatedehydrogenase, see Aranov, A. M. et al, J. Med. Chem., 41(24),4790-4799, 1998). A particularly promising area of research hasidentified the role of cysteine proteases, encoded by the parasite, thatplay a pivotal role during the life cycle of the parasite (McKerrow, J.H., et al, Bioorg. Med. Chem., 7, 639-644, 1999). Proteases form asubstantial group of biological molecules which to date constituteapproximately 2% of all the gene products identified following analysisof several genome sequencing programmes (e.g. see Southan, C. J. Pept.Sci, 6, 453-458, 2000). Proteases have evolved to participate in anenormous range of biological processes, mediating their effect bycleavage of peptide amide bonds within the myriad of proteins found innature. This hydrolytic action is performed by initially recognising,then binding to, particular three-dimensional electronic surfacesdisplayed by a protein, which aligns the bond for cleavage preciselywithin the protease catalytic site. Catalytic hydrolysis then commencesthrough nucleophilic attack of the amide bond to be cleaved either viaan amino acid side-chain of the protease itself, or through the actionof a water molecule that is bound to and activated by the protease.Proteases in which the attacking nucleophile is the thiol side-chain ofa Cys residue are known as cysteine proteases. The generalclassification of ‘cysteine protease’ contains many members found acrossa wide range of organisms from viruses, bacteria, protozoa, plants andfungi to mammals.

Biological investigation of Trypanosoma cruzi infection has highlighteda number of specific enzymes that are crucial for the progression of theparasite's life cycle. One such enzyme, cruzipain, a cathepsin L-likecysteine protease, is a clear therapeutic target for the treatment ofChagas' disease ((a) Cazzulo, J. J. et al, Curr. Pharm. Des. 7,1143-1156, 2001; (b) Caffrey, C. R. et al, Curr. Drug Targets, 1,155-162, 2000). Although the precise biological role of cruzipain withinthe parasite's life cycle remains unclear, elevated cruzipain messengerRNA levels in the epimastigote developmental stage indicate a role inthe nutritional degradation of host-molecules in lysosomal-like vesicles(Engel, J. C. et al, J. Cell. Sci, 111, 597-606, 1998). The validationof cruzipain as a viable therapeutic target has been achieved withincreasing levels of complexity. Addition of a general cysteine proteaseinhibitor, Z-Phe-Ala-FMK to Trypanosoma cruzi-infected mammalian cellcultures blocked replication and differentiation of the parasite, thusarresting the parasite life cycle (Harth, G., et al, Mol. Biochem.Parasitol. 58, 17-24, 1993). Administration of a vinyl sulphone-basedinhibitor in a Trypanosoma cruzi-infected murine animal model not onlyrescued the mice from lethal infections, but also produced a completerecovery (Engel, J. C. et al, J. Exp. Med, 188(4), 725-734, 1998).Numerous other in vivo studies have confirmed that infections withalternative parasites such as Leishmania major (Selzer, P. M. et al,Proc. Nat'l. Acad. Sci. U.S.A., 96, 11015-11022, 1999), Schistosomamansoni and Plasmodium falciparium (Olson, J. E. et al, Bioorg. Med.Chem., 7, 633-638, 1999) can be halted or cured by treatment withcysteine protease inhibitors.

A variety of synthetic approaches have been described towards the designof cruzipain inhibitors. However, although providing a biological‘proof-of-principle’ for the treatment of Trypanosoma cruzi infection,current inhibitors exhibit a number of biochemical and physicalproperties that may preclude their clinical development. (e.g. see (a)Brinen, L. S. et al, Structure, 8, 831-840, 2000, peptidomimetic vinylsulphones, possible adverse mammalian cell toxicity (see McKerrow, J. H.and Engel, J. unpublished results cited in Scheidt, K. A. et al, Bioorg.Med. Chem, 6, 2477-2494, 1998); (b) Du, X. et al, Chem. Biol., 7,733-742, 2000, aryl ureas, generally with low μM activity, and highClogP values, thus poor aqueous solubility and probably low oralbioavailability; (c) Roush, W. R. et al, Tetrahedron, 56, 9747-9762,2000, peptidyl epoxysuccinates, irreversible inhibitors, with potentactivity verses house-keeping mammalian proteases such as cathepsin B;(d) Li, R. et al, Bioorg. Med. Chem. 4(9), 1421-1427, 1996,bisarylacylhydrazides and chalcones, polyhydroxylated aromatics; (e)U.S. Pat. No. 6,143,931, WO 9846559, non-peptide α-ketoamides). Of themany different approaches to enzyme inhibition to date, only thecruzipain protease inhibitors have proven effective in curingdisease-related animal models of Trypanosoma cruzi infection. Therefore,a clear medical need exists to progress these ‘proof-of-principle’findings towards clinical candidates, suitable for human use, throughthe discovery of more efficacious cruzipain inhibitors that have adesirable combination of potency, selectivity, low toxicity andoptimised pharmacokinetic parameters.

Cruzipain and indeed many other crucial parasitic proteases belong tothe papain-like CA C1 family and have close structural mammalianhomologues. Cysteine proteases are classified into ‘clans’ based upon asimilarity in the three-dimensional structure or a conserved arrangementof catalytic residues within the protease primary sequence.Additionally, ‘clans’ are further classified into ‘families’ in whicheach protease shares a statistically significant relationship with othermembers when comparing the portions of amino acid sequence whichconstitute the parts responsible for the protease activity (see Barrett,A. J et al, in ‘Handbook of Proteolytic Enzymes’, Eds. Barrett, A. J.,Rawlings, N. D., and Woessner, J. F. Publ. Academic Press, 1998, for athorough discussion). To date, cysteine proteases have been classifiedinto five clans, CA, CB, CC, CD and CE (Barrett, A. J. et al, 1998). Aprotease from the tropical papaya fruit ‘papain’ forms the foundation ofclan CA, which currently contains over 80 distinct and complete entriesin various sequence databases, with many more expected from the currentgenome sequencing efforts. Proteases of clan CA/family C1 have beenimplicated in a multitude of disease processes e.g. human proteases suchas cathepsin K (osteoporosis), cathepsin S (autoimmune disorders),cathepsin L (metastases) or parasitic proteases such as falcipain(malaria parasite Plasmodium falciparum), cruzipain (Trypanosoma cruziinfection). Recently a bacterial protease, staphylopain (S. aureusinfection) has also been tentatively assigned to clan CA. X-raycrystallographic structures are available for a range of the abovementioned proteases in complex with a range of inhibitors e.g. papain(PDB entries, 1pad, 1pe6, 1pip, 1pop, 4pad, 5pad, 6pad, 1ppp, 1the,1csb, 1huc), cathepsin K (1au0, 1au2, 1au3, 1au4, 1atk, 1mem, 1bgo,1ayw, 1ayu), cathepsin L (1cs8), cathepsin S (currently on-hold, butpublished McGrath, M. E. et al, Protein Science, 7, 1294-1302, 1998),cretin (a recombinant form of cruzipain see Eakin, A. E. et al, 268(9),6115-6118, 1993) (1ewp, 1aim, 2aim, 1F29, 1F2A, 1F2B, 1F2C),staphylopain (1cv8). Each of the structures displays a similar overallactive-site topology, as would be expected by their ‘clan’ and ‘family’classification and such structural similarity exemplifies one aspect ofthe difficulties involved in discovering a selective inhibitor ofcruzipain suitable for human use. However, subtle differences in termsof the depth and intricate shape of the active site groove of each CA C1protease are evident, which may be exploited for selective inhibitordesign. Additionally, many of the current substrate-based inhibitorcomplexes of CA C1 family proteases show a series of conserved hydrogenbonds between the inhibitor and the protease backbone, which contributesignificantly to inhibitor potency. Primarily a bidentate hydrogen-bondis observed between the protease Gly66 (C═O)/inhibitor N—H and theprotease Gly66(NH)/inhibitor (C═O), where the inhibitor (C═O) and (NH)are provided by an amino acid residue NHCHRCO that constitutes the S2sub-site binding element within the inhibitor (see Berger, A. andSchecter, I. Philos. Trans. R. Soc. Lond. [Biol.], 257, 249-264, 1970for a description of protease binding site nomenclature). A furtherhydrogen-bond between the protease main-chain (C═O) of asparagine oraspartic acid (158 to 163, residue number varies between proteases) andan inhibitor (N—H) is often observed, where the inhibitor (N—H) isprovided by the S1 sub-site binding element within the inhibitor. Thus,the motif X—NHCHRCO—NH—Y is widely observed amongst the prior artsubstrate-based inhibitors of CA C1 proteases.

In the prior art, the development of cysteine protease inhibitors forhuman use has recently been an area of intense activity. Considering theCA C1 family members, particular emphasis has been placed upon thedevelopment of inhibitors of human cathepsins, primarily cathepsin K(osteoporosis), cathepsin S (autoimmune disorders) and cathepsin L(metastases), through the use of peptide and peptidomimetic nitrites(e.g. see WO-A-0109910, WO-A-0051998, WO-A-0119816, WO-A-9924460,WO-A-0049008, WO-A-0048992, WO-A-0049007, WO-A-0130772, WO-A-0055125,WO-A-0055126, WO-A-0119808, WO-A-0149288, WO-A-0147886), linear andcyclic peptide and peptidomimetic ketones (e.g. see Veber, D. F. andThompson, S. K., Curr. Opin. Drug Discovery Dev., 3(4), 362-369, 2000,WO-A-0170232, WO-A-0178734, WO-A-0009653, WO-A-0069855, WO-A-0029408,WO-A-0134153 to WO-A-0134160, WO-A-0029408, WO-A-9964399, WO-A-9805336,WO-A-9850533), ketoheterocycles (e.g. see WO-A-0055144, WO-A-0055124)and monobactams (e.g. see WO-A-0059881, WO-A-9948911, WO-A-0109169). Theprior art describes potent in vitro inhibitors, but also highlights themany difficulties in developing a human therapeutic. For example,WO-A-9850533 and WO-A-0029408 describe compounds that may be referred toas cyclic ketones and are inhibitors of cysteine proteases with aparticular reference towards papain family proteases and as a mostpreferred embodiment, cathepsin K. WO-A-9850533 describes compoundssubsequently detailed in the literature as potent inhibitors ofcathepsin K with good oral bioavailability (Witherington, J.,‘Tetrahydrofurans as Selective Cathepsin K Inhibitors’, RSC meeting,Burlington House, London, 1999). The compounds of WO-A-9850533 werereported to bind to cathepsin K through the formation of a reversiblecovalent bond between the tetrahydrofuran carbonyl and the active sitecatalytic cysteine residue (Witherington, J., 1999). Additionally, thesame cyclic ketone compounds are described in WO-A-9953039 as part of awide-ranging description of inhibitors of cysteine proteases associatedwith parasitic diseases, with particular reference to the treatment ofmalaria by inhibition of falcipain. However, subsequent literaturedescribes the cyclic ketone compounds of WO-A-9850533 to be unsuitablefor further development or for full pharmacokinetic evaluation due to aphysiochemical property of the inhibitors, the poor chiral stability ofthe α-aminoketone chiral centre (Marquis, R. W. et al, J. Med. Chem.,44(5), 725-736, 2001). WO-A-0069855 describes compounds that may also bereferred to as cyclic ketones with particular reference towardsinhibition of cathepsin S. The compounds of WO-A-0069855 are consideredto be an advance on compounds of WO-A-9850533 due to the presence of theβ-substituent on the cyclic ketone ring system that provides increasedchiral stability to the α-carbon of the cyclic ketone ring system. In anattempt to solve the problem of poor chiral integrity, subsequentliterature has provided a closely related cyclic ketone series to thatdescribed in WO-A-9850533, where an approximately 300-fold loss ininhibitor potency was observed upon introduction of an alkyl group inplace of the labile α-proton (Marquis, R. W. et al, J. Med. Chem., 44,1380-1395, 2001). Additionally, subsequent literature has shown thatwithin the cyclic ketone series described in WO-A-9850533, the α-(S)isomer is approximately 10 to 80-fold more potent than the α-(R) isomer(Fenwick, A. E. et al, Bioorg. Med. Chem. Lett., 11, 199-202, 2001).

It has now been discovered that certain compounds, defined by generalformula (I), are potent and selective cruzipain protease inhibitorswhich are useful in the treatment of Trypanosoma cruzi infection. Othercompounds defined by general formula (I) are protease inhibitors acrossa broad range of CA C1 cysteine proteases and compounds useful in thetreatment of diseases caused by cysteine proteases. Compounds describedby general formula (I) contain an α-alkyl group, of theR-stereo-configuration (or the S-stereo-configuration when Z=‘S’), yetsurprisingly compounds defined by general formula (I) retain goodpotency. The present invention provides substituted (3aR,6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide, (3aS,6aR)N-(3-oxo-hexahydrocyclopenta[b]thiophene-3a-yl)alkylamide, (3aR,6aS)N-(3-oxo-hexahydropentalen-3a-yl)alkylamide or (3aR,6aR)N-(3-oxo-hexahydrocyclo penta[b]pyrrol-3a-yl)alkylamide compoundsdefined by general formula (I).

Accordingly, the first aspect of the invention provides a compoundaccording to general formula (I):

wherein: R¹=C₀₋₇-alkyl (when C=0, R¹ is simply hydrogen),C₃₋₆-cycloalkyl or —Ar—C₀₋₇-alkyl (when C=0, R¹ is simply an aromaticmoiety Ar);

-   -   Z=O, S, CR²R³ or NR⁴, where R⁴ is chosen from C₀₋₇-alkyl,        C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl;    -   Each of R² and R³ is independently chosen from C₀₋₇-alkyl,        C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl, O—C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl,        O—Ar—C₀₋₇-alkyl S—C₀₋₇-alkyl, S—C₃₋₆-cycloalkyl,        S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-alkyl, NH—C₃₋₆-cycloalkyl,        NH—Ar—C₀₋₇-alkyl, N(C₀₋₇-alkyl)₂, N(C₃₋₆-cycloalkyl)₂ or        N(Ar—C₀₋₇-alkyl)₂;    -   Y=CR⁵R⁶—CO, where R⁵, R⁶ are chosen from C₀₋₇-alkyl,        C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl;    -   (X)_(o)=CR⁷R⁸, where R⁷ and R⁸ are independently chosen from        C₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and o is a number        from zero to three;    -   (W)_(n)=O, S, C(O), S(O) or S(O)₂ or NR⁹, where R⁹ is chosen        from C₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and n is zero        or one;    -   (V)_(m)=C(O), C(S), S(O), S(O)₂, S(O)₂NH, OC(O), NHC(O), NHS(O),        NHS(O)₂, OC(O)NH, C(O)NH or CR¹⁰R¹¹, where R¹⁰ and R¹¹ are        independently chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl,        Ar—C₀₋₇-alkyl and m is a number from zero to three, provided        that when m is greater than one, (V)_(m) contains a maximum of        one carbonyl or sulphonyl group;    -   U=a stable 5- to 7-membered monocyclic or a stable 8- to        11-membered bicyclic ring which is either saturated or        unsaturated and which includes zero to four heteroatoms (as        detailed below):    -   wherein R¹² is:        -   C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl, O—C₀₋₇-alkyl,            O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl, S—C₀₋₇-alkyl,            S—C₃₋₆-cycloalkyl, S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-alkyl,            NH—C₃₋₆-cycloalkyl, NH—Ar—C₀₋₇-alkyl, N(C₀₋₇-alkyl)₂,            N(C₃₋₆-cycloalkyl)₂ or N(Ar—C₀₋₇-alkyl)₂ or, when it is part            of the group CHR¹² or CR¹², R¹² may be halogen;        -   A is chosen from:            -   CH₂, CHR¹², O, S and NR¹³;            -   wherein R¹² is as defined above and R¹³ is chosen from:                -   C₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl;        -   B, D and G are independently chosen from:            -   CR¹², where R¹² is as defined above, or N;        -   E is chosen from:            -   CH₂, CHR¹², O, S and NR¹³, where R¹² and R¹³ are defined                as above;        -   J, L, M, R, T, T₂, T₃ and T₄ are independently chosen from:            -   CR¹² and N, where R¹² is as defined above;        -   T₅ is chosen from:            -   CH or N;        -   q is a number from one to three, thereby defining a 5-, 6-            or 7-membered ring.

B, D, G, J, L, M, R, T, T₂, T₃ and T₄ may additionally represent anN-oxide (N→O).

The present invention includes all salts, hydrates, solvates, complexesand prodrugs of the compounds of this invention. The term “compound” isintended to include all such salts, hydrates, solvates, complexes andprodrugs, unless the context requires otherwise.

Appropriate pharmaceutically and veterinarily acceptable salts of thecompounds of general formula (I) include salts of organic acids,especially carboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate,digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate,heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate,3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate, organicsulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids. Salts which are not pharmaceutically orveterinarily acceptable may still be valuable as intermediates.

Prodrugs are any covalently bonded compounds which release the activeparent drug according to general formula (I) in vivo. A prodrug may forexample constitute ketal or hemiketal derivative of the exocyclc ketonefunctionality present in the (3aR,6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide, (3aS,6aR)N-(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide, (3aR, 6aS)N-(3-oxo-hexahydropentalen-3a-yl)alkylamide or (3aR, 6aR)N-(3-oxo-hexahydrocyclo penta[b]pyrrol-3a-yl)alkylamide scaffold. If achiral centre or another form of isomeric centre is present in acompound of the present invention, all forms of such isomer or isomers,including-enantiomers and diastereoisomers, are intended to be coveredherein. Compounds of the invention containing a chiral centre may beused as a racemic mixture, an enantiomerically enriched mixture, or theracemic mixture may be separated using well-known techniques and anindividual enantiomer may be used alone.

‘Halogen’ as applied herein is meant to include F, Cl, Br, I;

‘Heteroatom’ as applied herein is meant to include O, S and N;

‘C₀₋₇-alkyl’ as applied herein is meant to include stable straight andbranched chain aliphatic carbon chains containing zero (i.e. simplyhydrogen) to seven carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyland any simple isomers thereof. Additionally, any C₀₋₇-alkyl mayoptionally be substituted at any point by one, two or three halogenatoms (as defined above) for example to give a trifluoromethylsubstituent. Furthermore, C₀₋₇-alkyl may contain one or more heteroatoms(as defined above) for example to give ethers, thioethers, sulphones,sulphonamides, substituted amines, amidines, guanidines, carboxylicacids, carboxamides. If the heteroatom is located at a chain terminusthen it is appropriately substituted with one or two hydrogen atoms. Aheteroatom or halogen is only present when C₀₋₇-alkyl contains a minimumof one carbon atom.

C₁₋₇-alkyl as applied herein is meant to include the definitions forC₀₋₇-alkyl (as defined above) but describes a substituent that comprisesa minimum of one carbon.

‘C₃₋₆-cycloalkyl’ as applied herein is meant to include any variation of‘C₀₋₇-alkyl’ which additionally contains a carbocyclic ring such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The carbocyclic ringmay optionally be substituted with one or more halogens (as definedabove) or heteroatoms (as defined above) for example to give atetrahydrofuran, pyrrolidine, piperidine, piperazine or morpholinesubstituent.

‘Ar—C₀₋₇-alkyl’ as applied herein is meant to include any variation ofC₀₋₇-alkyl which additionally contains an aromatic ring moiety ‘Ar’. Thearomatic ring moiety Ar can be a stable 5 or 6-membered monocyclic or astable 9 or 10 membered bicyclic ring which is unsaturated, as definedpreviously for U in general formula (I). The aromatic ring moiety Ar maybe substituted by R¹² (as defined above for U in general formula (I)).When C=0 in the substituent Ar—C₀₋₇-alkyl, the substituent is simply thearomatic ring moiety Ar.

Other expressions containing terms such as alkyl and cycloalkyl areintended to be construed according to the definitions above. For example“C₁₋₄ alkyl” is the same as C₀₋₇-alkyl except that it contains from oneto four carbon atoms.

If different structural isomers are present, and/or one or more chiralcentres are present, all isomeric forms are intended to be covered.Enantiomers are characterised by the absolute configuration of theirchiral centres and described by the R- and S-sequencing rules of Cahn,Ingold and Prelog. Such conventions are well known in the art (e.g. see‘Advanced Organic Chemistry’, 3^(rd) edition, ed. March, J., John Wileyand Sons, New York, 1985).

Preferred compounds of general formula (I) include, but are not limitedto, those in which, independently or in combination:

Z is O, S, NH or CH₂.

Additionally, preferred compounds of general formula (I) include, butare not limited to, those in which, independently or in combination

Z is NR⁴, where R⁴ is Ar—C₁₋₄-alkyl or a substituted carbonyl orsulphonyl group

Thus, examples of preferred compounds include those containing a (3aR,6aR) N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide, (3aS,6aR)N-(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide, (3aR,6aS)N-(3-oxo-hexahydropentalen-3a-yl)alkylamide or a (3aR,6aR)N-(3-oxo-hexahydrocyclopenta[b]pyrrol-3a-yl)alkylamide bicyclic moietyas shown below.

(3aR, 6aR)-N-(3-Oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide

(3aS, 6aR)-N-(3-Oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide

(3aR, 6aS)-N-(3-Oxo-hexahydropentalen-3a-yl)alkylamide

(3aR, 6aR)-N-(3-Oxo-hexahydrocyclopenta[b]pyrrol-3a-yl)alkylamide

In compounds of general formula (I), it is preferred that R¹ comprisesC₀₋₇-alkyl or Ar—C₀₋₇-alkyl. Thus, for example, preferred R¹ moietiesinclude hydrogen, or a straight or branched alkyl chain, or a straightor branched heteroalkyl chain, or an optionally substituted arylalkylchain, or an optionally substituted arylheteroalkyl chain.

It is particularly preferred that R¹ is hydrogen or C₁₋₄ alkyl orAr—C₁₋₄-alkyl and examples of such R¹ substituents include, but are notlimited to:

where R¹² is defined above.

In preferred compounds of general formula (I), Y is CHR⁶CO where R⁶ isselected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl, for example hydrogen, astraight or branched alkyl chain, a straight or branched heteroalkylchain, an optionally substituted arylalkyl chain or an optionallysubstituted arylheteroalkyl chain. Additionally, in preferred compoundsof general formula (I), R⁶ is selected from C₃₋₆-cycloalkyl, for examplecyclohexylmethyl.

Examples of preferred Y substituents include the following:

wherein R¹², R¹³ and Ar are as defined above.

More preferred R⁶ groups include C₁₋₄-alkyl, which may be substitutedwith OH, NR¹³R¹³, COOR¹³, or CONR¹³ or cycloalkylmethyl orAr—C₁₋₄-alkyl, where the aryl group may be substituted with R¹²; whereineach R¹² and R¹³ is independently as defined above.

Even more preferred R⁶ groups comprise Ar—CH₂—, where the aromatic ringis an optionally substituted phenyl or monocyclic heterocycle.Additionally, even more preferred R⁶ groups comprise simple branchedalkyl groups such as isobutyl or straight heteroalkyl chains such asbenzysulfanylmethyl or benzylsulphonylmethyl. Furthermore, even morepreferred R⁶ groups comprise cyclohexylmethyl. Examples of even morepreferred Y substituents comprise the following,

wherein R¹² and Ar are as defined previously

It is preferred that in the group (X)_(o), each of R⁷ and R⁸ is selectedfrom C₀₋₇-alkyl or Ar—C₀₋₇-alkyl, for example hydrogen, a straight orbranched alkyl chain, a straight or branched heteroalkyl chain, anoptionally substituted arylalkyl chain or an optionally substitutedarylheteroalkyl chain.

More preferred (X)_(o) groups comprise R⁷ chosen from hydrogen; R⁸ isC₁₋₄-alkyl, which may be substituted with OH, NR¹³R¹³, COOR¹³, orCONR¹³; or Ar—C₁₋₄-alkyl, where the aryl group may be substituted withR¹², wherein each R¹² and R¹³ is independently as defined above.

Examples of preferred (X)_(o) groups include the following:

wherein R¹² and R¹³ are as defined previously.

Even more preferred compounds of general formula (I), comprise (X)_(o)groups that are simple alkyl groups such as methylene and where o=0 or1.

In the group (W)_(n), W is preferably O, S, SO₂, S(O), C(O) or NR⁹,where R⁹ is C₀₋₄-alkyl; and n is 0 or 1.

More preferred compounds of general formula (I), comprise (W)_(n) groupsdefined as O, S, SO₂, C(O) and NH where n=0 or 1.

Yet even more preferred compounds of general formula (I), comprise(W)_(n) groups defined as NH where n=1.

In the group (V)_(m):

-   -   V is preferably C(O), C(O)NH or CHR¹¹, where R¹¹ is C₀₋₄-alkyl;        and    -   m is 0 or 1.

Preferred V and W substituent combinations encompassed by generalformula (I) include, but are not limited to:

Additionally, a preferred V and W substituent combination encompassed bygeneral formula (I) includes, but is not limited to:

More preferred V, W and X substituent combinations encompassed bygeneral formula (I) comprise, but are not limited to

In preferred compounds of general formula (I), U comprises an optionallysubstituted 5- or 6-membered saturated or unsaturated heterocycle or Argroup or an optionally substituted saturated or unsaturated 9- or10-membered heterocycle or Ar group. Examples of such preferred U ringsinclude the following:

and also the following

wherein R¹² is as defined previously.

More preferred compounds of general formula (I), contain a U groupcomprising of a bulky alkyl or aryl group at the para position of anaryl Ar. Also, more preferred compounds contain a meta or para-biarylAr—Ar, where Ar is as previously defined. Additionally, more preferredcompounds contain a 6,6 or 6,5 or 5,6-fused aromatic ring. Examples ofmore preferred U groups are

wherein R¹², D, E, G, J, L, M, R, T, T₂, T₃ and T₄ are as definedpreviously.

Even more preferred compounds of general formula (I), particularly forinhibition of cruzipain, contain a U group comprising a 6-membered Arring containing a bulky alkyl or aryl group at the para position, whereAr is as previously defined

wherein R¹², D, E, G, J, L, M, R and T are as defined previously

Yet even more preferred compounds of general formula (I), contain a Ugroup comprising but are not limited to the following,

wherein R¹², D, E, G, J and L are as defined previously.

Abbreviations and symbols commonly used in the peptide and chemical artsare used herein to describe compounds of the present invention,following the general guidelines presented by the IUPAC-IUB JointCommission on Biochemical Nomenclature as described in Eur. J. Biochem.,158, 9-, 1984. Compounds of formula (I) and the intermediates andstaring materials used in their preparation are named in accordance withthe IUPAC rules of nomenclature in which the characteristic groups havedecreasing priority for citation as the principle group. An examplecompound of formula (I), compound (1) in which R¹ is H, Z is oxygen, Yis 4-methylpentyl, (X)₀ is zero, (W)_(n) is oxygen, (V)_(m) is methyleneand U is phenyl is thus named:

(3aR, 6aR) 2-Benzyloxy-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-amide

A second example compound of formula (I), compound (2) in which R¹ is H,Z is sulphur, Y is 4-methylpentyl, (X)₀ is zero, (W)_(n) is oxygen,(V)_(m) is methylene and U is phenyl is thus named:

(3aS, 6aR) 2-Benzyloxy-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)-amide

A third example compound of formula (I), compound (3) in which R¹ is H,Z is methylene, Y is 4-methylpentyl, (X)₀ is zero, (W)_(n) is oxygen,(V)_(m) is methylene and U is phenyl is thus named:

(3aR, 6aS) 2-Benzyloxy-4-methyl-pentanoic acid(3-oxo-hexahydro-pentalen-3a-yl)-amide

A fourth example compound of formula (I), compound (4) in which R¹ is H,Z is NH, Y is 4-methylpentyl, (X)₀ is zero, (W)_(n) is oxygen, (V)_(m)is methylene and U is phenyl is thus named:

(3aR, 6aR) 2-Benzyloxy-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]pyrrol-3a-yl)-amide

Compounds of the invention include, but are not limited to, thefollowing examples that are the (3aR, 6aR) isomer of general formula (I)where Z=‘O’ and R¹=‘H’, and also include the equivalent analoguesincluded in the full definition of Z and R¹

4-tert-Butyl-N-[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]-4-isopropyl-benzamide

4-Difluoromethoxy-N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Naphthalene-1-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Quinoline-6-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-3-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzothiazole-5-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Biphenyl-4-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-imidazol-1-yl-benzamide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-thiophen-2-ylbenzamide

N-[2-(4-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-Phenylthiophene-2-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Thiophen-2-ylthiazole-4-carboxylic acid[2-4-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

3-Phenylpyrrole-1-carboxylic acid[2-(4-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-tert-Butyl-N-[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-benzamide

N-[3-Methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-benzamide

4-Isopropyl-N-[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-benzamide

4-Difluoromethoxy-N-[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-benzamide

N-[3-Methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Naphthalene-1-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Quinoline-6-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Benzo[b]thiophene-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Benzo[b]thiophene-3-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Benzothiazole-5-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Biphenyl-4-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

N-[3-Methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-4-imidazol-1-ylbenzamide

N-[3-Methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-4-thiophen-2-ylbenzamide

N-[3-Methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

4-Phenylthiophene-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

2-Thiophen-2-ylthiazole-4-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

3-Phenylpyrrole-1-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

4-tert-Butyl-N-[2-3-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]-4-isopropyl-benzamide

4-Difluoromethoxy-N-[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Naphthalene-1-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Quinoline-6-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-3-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzothiazole-5-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Biphenyl-4-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-imidazol-1-yl-benzamide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-thiophen-2-ylbenzamide

N-[2-(3-Hydroxyphenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-Phenylthiophene-2-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Thiophen-2-ylthiazole-4-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

3-Phenylpyrrole-1-carboxylic acid[2-(3-hydroxyphenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-tert-Butyl-N-[2-(4-fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[2-(4-fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]-4-isopropyl-benzamide

4-Difluoromethoxy-N-[2-(4-fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Naphthalene-1-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Quinoline-6-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-3-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzothiazole-5-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Biphenyl-4-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

N-[2-(4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-imidazol-1-yl-benzamide

N-[2-(4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-thiophen-2-ylbenzamide

N-[2-(4-Fluorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-Phenylthiophene-2-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Thiophen-2-ylthiazole-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

3-Phenylpyrrole-1-carboxylic acid[2-(4-fluorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-tert-Butyl-N-[2-(4-chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[2-(4-chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]-4-isopropyl-benzamide

4-Difluoromethoxy-N-[2-(4-chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Naphthalene-1-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Quinoline-6-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-3-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzothiazole-5-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Biphenyl-4-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-imidazol-1-yl-benzamide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-thiophen-2-ylbenzamide

N-[2-(4-Chlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-Phenylthiophene-2-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Thiophen-2-ylthiazole-4-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

3-Phenylpyrrole-1-carboxylic acid[2-(4-chlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-tert-Butyl-N-[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethoxy-benzamide

4-Dimethylamino-N-[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]-4-isopropyl-benzamide

4-Difluoromethoxy-N-[2-3,4-dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-benzamide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-trifluoromethyl-benzamide

Naphthalene-2-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Naphthalene-1-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Quinoline-6-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-3-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzothiazole-5-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Biphenyl-4-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-imidazol-1-yl-benzamide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-4-thiophen-2-ylbenzamide

N-[2-(3,4-Dichlorophenyl)-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-5-thiophen-2-ylnicotinamide

2-Phenylthiazole-4-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Pyridin-3-ylthiazole-4-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

4-Phenylthiophene-2-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-Thiophen-2-ylthiazole-4-carboxylic acid[2-(3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

3-Phenylpyrrole-1-carboxylic acid[2-3,4-dichlorophenyl)-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

2-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoic acid(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

2-(4-tert-Butyl-benzylsulfanyl)-3-(4-hydroxyphenyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

2-(4-tert-Butyl-benzylsulfanyl)-3-(3-hydroxyphenyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

2-(4-tert-Butyl-benzylsulfanyl)-3-(4-fluorophenyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

2-(4-Hydroxybenzyl)-4-oxo-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-4-(3-phenyl-pyrrol-1-yl)-butyramide

2-(4-Hydroxybenzyl)-4-oxo-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-4-(3-phenyl-pyrrolidine-1-yl)-butyramide

4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrol-1-yl)-ethyl]-pentanoic acid(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrolidin-1-yl)-ethyl]-pentanoic acid(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

2-[2-(1,3-Dihydro-isoindol-2-yl)-2-oxo-ethyl]-4-methyl-pentanoic acid(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

4-(1,3-Dihydro-isoindol-2-yl)-2-(4-hydroxybenzyl)-4-oxo-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-butyramide

4-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-(4-hydroxybenzyl)-4-oxo-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-butyramide

2-[2-(3,4Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicacid (3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

Additional compounds of the invention include, but are not limited to,the following examples that are the (3aR, 6aR) isomer of general formula(I) where Z=‘O’ and R¹=‘H’, and also include the equivalent analoguesincluded in the full definition of Z and R¹

Furan-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Furan-3-carboxylic acid[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Thiophene-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Thiophene-3-carboxylic acid[3-methyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Benzo[b]thiophene-2-carboxylic acid[3-methyl-1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Furan-2-carboxylic acid[2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Furan-3-carboxylic acid[2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Thiophene-2-carboxylic acid [[2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Thiophene-3-carboxylic acid [2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Furan-2-carboxylic acid[3,3-dimethyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Furan-3-carboxylic acid[3,3-dimethyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Thiophene-2-carboxylic acid[3,3-dimethyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Thiophene-3-carboxylic acid[3,3-dimethyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Benzo[b]thiophene-2-carboxylic acid[3,3-dimethyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-butyl]-amide

Furan-2-carboxylic acid[2-benzylsulfanyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Furan-3-carboxylic acid[2-benzylsulfanyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Thiophene-2-carboxylic acid [2-benzylsulfanyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Thiophene-3-carboxylic acid [2-benzylsulfanyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[2-benzylsulfanyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]-amide

Furan-2-carboxylic acid[1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide

Furan-3-carboxylic acid(1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide

Thiophene-2-carboxylic acid[1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide

Thiophene-3-carboxylic acid[1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide

Benzo[b]thiophene-2-carboxylic acid[1-(3-oxo-hexahydro-cyclopenta[b]furan-3a-ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide

2-Benzyloxy-3-cyclohexyl-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-propionamide

Morpholine-4-carboxylic acid2-cyclohexyl-1-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethylester

2-Cyclohexylmethyl-4-morpholin-4-yl-4-oxo-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-butyramide

2-Biphenyl-3-yl-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-amide

3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

3-Cyclohexyl-2-(furan-3-ylmethylsulfanyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

3-Cyclohexyl-2-(furan-3-ylmethanesulfonyl)-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-propionamide

2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-amide

2-(4-tert-Butyl-benzyloxy)-4-methyl-pentanoic acid(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-amide

Considering all of the above examples, it is also intended to includethe oxidised analogues of capping groups that contain a readily oxidisednitrogen to give the N-oxide or a readily oxidised sulphur to give thesulphone. The following structures are illustrative examples;

To those skilled in the practices of organic chemistry, compounds ofgeneral formula (I) may be readily synthesised by a number of chemicalstrategies, performed either in solution or on the solid phase (seeAtherton, E. and Sheppard, R. C. In ‘Solid Phase Peptide Synthesis: APractical Approach’, Oxford University Press, Oxford, U.K. 1989, for ageneral review of solid phase synthesis principles). The solid phasestrategy is attractive in being able to generate many thousands ofanalogues, typically on a 5-100 mg scale, through established parallelsynthesis methodologies (e.g. see (a) Bastos, M.; Maeji, N. J.; Abeles,R. H. Proc. Natl. Acad. Sci. USA, 92, 6738-6742, 1995).

Therefore, one strategy for the synthesis of compounds of generalformula (I) comprises:

-   (a) Preparation of an appropriately functionalised and protected    cyclopentane bicyclic ketone building block in solution.-   (b) Attachment of the building block (a) to the solid phase through    a linker that is stable to the conditions of synthesis, but readily    labile to cleavage at the end of a synthesis (see James, I. W.,    Tetrahedron, 55(Report N ^(o) 489), 4855-4946, 1999, for examples of    the ‘linker’ function as applied to solid phase synthesis).-   (c) Solid phase organic chemistry (see Brown, R. D. J. Chem. Soc.,    Perkin Trans.1, 19, 3293-3320, 1998), to construct the remainder of    the molecule.-   (d) Compound cleavage from the solid phase into solution.-   (e) Cleavage work-up and compound analysis.

The first stage in a synthesis of compounds of general formula (I) isthe preparation in solution of a functionalised and protected buildingblock. A typical scheme towards the (3aR,6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide (8) is detailed inScheme 1.

FmOC(O) denotes the well known amine protecting group 9-fluorenylmethoxycarbonyl (Fmoc, see Atherton, E. and Sheppard, R. C., 1989) and‘Pg’ denotes either a free hydroxyl or an hydroxyl protecting group suchas tert-butyl ether. In the illustrated case, condensation withdiazomethane provides R¹=H.

Considering step (a), synthesis commences from a suitably protected1-amino-2-hydroxycyclopentanecarboxylic acid (5). The core aminoacid isaccessible through a variety of literature methods such as theasymmetric Strecker or Bucherer-Bergs syntheses e.g. (a) Ohfune, Y.,Nanba, K., Takada, I., Kan, T., Horikawa, N., Nakajima, T. Chirality, 9,459-462, 1997. (b) Ohfune, Y., Horikawa, N., J. Synth. Org. Chem Jpn.,55, 982-993, 1997. (c) Volk, F-J., Frahm, A. W. Liebigs Ann. Chem.1893-1903, 1996. (d) Fonderkar, K. P., Volk, F-J., Frahm, A. W.Tetrahedron: Asymmetry, 10, 727-735, 1999. Activation of the suitablyprotected 1-amino-2-hydroxycyclopentanecarboxylic acid (5) via isobutylchloroformate mixed anhydride, followed by condensation withdiazomethane, yields the diazomethylketone intermediate (7). Followingthe reaction conditions detailed in Scheme 1, formation of thediazoketone is clearly observed. However, an overall improvement inisolated yield of diazoketone (7) is obtained by pre-forming the acylfluoride of (5), which has a lesser propensity to form the poorly active(5R, 6R)6-alkoxy-2-(9H-fluoren-9-ylmethoxy)-3-oxa-1-azaapiro[4,4]non-1-en-4-one(6) and diazomethylketone intermediate (7) with lithium chloride inaqueous acetic acid provides the protected (3aR, 6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide (8). Introductionof simple R¹ substituents may be achieved by condensation of activated(5) with alternatives to diazomethane such as diazoethane (R¹=CH₃), or1-phenyloxydiazoethane (R¹=CH₂OPh).

The protected building blocks (synthesis exemplified by (3aR, 6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide (8) detailed inScheme 1 may be utilised in a solid phase synthesis of inhibitormolecules (steps (b) to (e)). Step (b), the solid phase linkage of analdehyde or ketone, has previously been described by a variety ofmethods (e.g. see (a) James, I. W., 1999, (b) Lee, A., Huang, L.,Ellman, J. A., J. Am. Chem. Soc, 121(43), 9907-9914, 1999, (c) Murphy,A. M., et al, J. Am. Chem. Soc, 114, 3156-3157, 1992). A suitable methodamenable to the reversible linkage of an alkyl ketone functionality suchas (8) is through a combination of the previously described chemistries.The semicarbazide, 4-[[(hydrazinocarbonyl) amino]methyl]cyclohexanecarboxylic acid. trifluoroacetate (9) (Murphy, A. M., et al, J. Am.Chem. Soc, 114, 3156-3157, 1992), may be utilised as illustrated inScheme 2, exemplified by linkage of the (3aR, 6aR)N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide (8).

Construct (10) is prepared through reaction of the linker molecule (9)and the (3aR, 6aR) N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide(8) by reflux in aqueous ethanol/sodium acetate. Although formation ofconstruct (10) is observed at 2 hr reaction, optimal formation ofconstruct (10) occurs at 24 hr reaction. Standard solid phase techniques(e.g. see Atherton, E. and Sheppard, R. C., 1989) are used to anchor theconstruct to an amino-functionalised solid phase through the freecarboxylic acid functionality of (10), providing the loaded construct(11). Acid mediated cleavage of the fully constructed compounds isoptimal at 24 hr reaction.

Loaded construct (11) may be reacted with a wide range of carboxylicacids available commercially or in the literature, to introduce theleft-hand portion ‘U—V—W—X—Y’ in general formula (I). In the simplestexample, the entire left hand portion of an inhibitor of general formula(I) comprises a capped aminoacid (Scheme 3), providing for exampleanalogues of general formula (I) where R⁵=‘H’, (X)_(o)=‘-’,(W)_(n)=‘NH’, R⁹=‘H’, n=1, (V)_(m)=‘CO’, m=1 and U=aryl

Alternatively, carboxylic acids can be prepared in solution bytraditional organic chemistry methods and coupled to construct (11) onthe solid phase (Schemes 4-8). For example (Scheme 4), treatment insolution of an amino acid, exemplified by (13) with sodiumnitrite/H₂SO₄, provides the α-hydroxyacid, exemplified by (14)(Degerbeck, F. et al, J. Chem. Soc, Perkin Trans. 1, 11-14, 1993).Treatment of α-hydroxyacid, (14) with sodium hydride in adimethylformamide/dichloromethane mixture followed by addition of benzylbromide, provides 2RS-benzyloxy-3-cyclohexylpropionic acid (15).Coupling of (15) to the solid phase construct (11) followed by cleavage,provides (16), an example of general formula (I) where R⁵=‘H’,(X)_(o)=‘-’, (W)_(n)=‘O’, n=1, (V)_(m)=‘CH₂’, i.e. R¹⁰, R¹¹=‘H’, m=1 andU=phenyl. To those skilled in the practices of organic synthesis, a widevariety of aminoacids such as (13) may be converted to the correspondingα-hydroxyacid such as (14) following the general conditions detailed.Additionally, benzylbromide may be replaced by any reasonableAr—CR¹⁰R¹¹-halide, providing many variations of carboxylic acid (15)following the general conditions detailed. In certain instances, it maybe advantageous to temporarily protect the carboxylic acid as the methylester (for example compound (21), Scheme 6) prior to reaction with thealkylhalide. The ester intermediate is then simply hydrolysed to acid(15). Analogues of (16), exploring a wide range of (V)_(m) and U ingeneral formula (I) may be prepared through the general conditionsdetailed in Scheme 4. Since the final synthetic step involves atrifluoroacetic acid (TFA) mediated cleavage of the solid phase boundcompound, analogues where the substituted ether is labile to TFA may beprepared in solution by an alternative route (see Scheme 11).

Alternatively, coupling of construct (11) (following removal of Fmoc)with the α-hydroxyacid (14), provides a versatile solid phase boundintermediate ‘Y’ substituent in general formula (I) that may be reactedwith many reagents. For example, the α-hydroxyl can be reacted underMitsunobu conditions (Hughes, D. L. Org. React.(N.Y), 42, 335-656, 1992)to give ethers (i.e. X=‘-’, W=‘O’, in general formula (I)) (seeGrabowska, U. et al, J. Comb. Chem., 2(5), 475-490, 2000, for an exampleof Mitsunobu reaction on the solid phase). Alternatively, the α-hydroxylcan be reacted with a carbamoyl chloride to give a carbamate (i.e.X=‘-’, W=‘O’, V=‘NHC(O)’, in general formula (I)).

Alternatively, (Scheme 5), treatment in solution of an amino acid,exemplified by (13) with sodium nitrite/H₂SO₄/potassium bromide providesthe α-bromoacid, exemplified by (17) (Souers, A. J. et al, Synthesis, 4,583-585, 1999) with retention of configuration. Treatment of α-bromoacid(17) with an alkylthiol exemplified by 4-tert-butylphenylmethanethiol(18) in diethylformamide/triethylamine, provides2S-(4-tert-butylbenzylsulfanyl)-4-methylpropionic acid (19), withinversion of configuration. Coupling of (19) to the solid phaseconstruct (11) followed by cleavage, provides (20), an example ofgeneral formula (I) where R⁵=‘H’, (X)_(o)=‘-’, (W)_(n)=‘S’, n=1,(V)_(m)=‘CH₂’, i.e. R¹⁰, R¹¹=‘H’, m=1 and U=4-tert-butylphenyl. To thoseskilled in the practices of organic synthesis, a wide variety ofaminoacids such as (13) may be converted to the correspondingα-bromoacid such as (17) following the general conditions detailed.Additionally, starting with the S-isomer of (13) gives the S-bromoacidanalogue of (17) and R-thioether analogue of (19). Additionally,(4-tert-butylphenyl)methanethiol (18) may be replaced by any reasonableAr—CR¹⁰R¹¹—SH, providing many variations of carboxylic acid (19)following the general conditions detailed. Thus analogues of (20)exploring a wide range of (V)_(m) and U in general formula (I) may beprepared through the general conditions detailed in Scheme 5.

Alternatively, coupling of construct (11) (following removal of Fmoc)with an α-bromoacid e.g. (17), provides a versatile intermediate ‘Y’substituent in general formula (I) that may be reacted with manyreagents. For example, the α-bromide can be displaced with nucleophilese.g. alcohols, thiols, carbanions etc, to give ethers (i.e. X=‘-’,W=‘O’, in general formula (I)), thioethers (i.e. X=‘-’, W=‘S’, ingeneral formula (I)). The thioethers may optionally be oxidised to thesulphone (see Scheme 9, i.e. X=‘-’, W=‘SO₂’, in general formula (I))(see Grabowska, U. et al, J. Comb. Chem., 2(5), 475-490, 2000, for anexample of bromide displacement and thioether oxidation on the solidphase).

Alternatively, (Scheme 6), treatment of an α-hydroxyacid, exemplified by(14) with trimethylsilylchloride and methanol provides the methyl ester(21). Activation of the free hydroxyl to the chloroformate with phosgenein dichloromethane followed by addition of morpholine, then hydrolysis,provides morpholine-4-carboxylic acid-1S-carboxy-2-cyclohexyl ethylester (22). Coupling of (22) to the solid phase construct (11) followedby cleavage, provides (23), an example of general formula (I) whereR⁵=‘H’, (X)_(o)=‘-’, (W)_(n)=‘O’, n=1, (V)_(m)=‘CO’ and U=morpholino. Tothose skilled in the practices of organic synthesis, a wide variety ofα-hydroxyacid esters such as (21) could be converted to the activatedchloroformate following the general conditions detailed. Additionally,morpholine may be replaced by any reasonable amine, providing manyvariations of carboxylic acid (22) following the general conditionsdetailed. Thus analogues of (23) exploring a wide range of (V)_(m) and Uin general formula (I) may be prepared through the general conditionsdetailed in Scheme 6.

Alternatively, (Scheme 7), a wide range of alkylsuccinate estersexemplified by 2R-cyclohexylmethylsuccinic acid 1-methyl ester (24) arecommercially available or readily prepared by known methods (see (a)Azam et al, J. Chem. Soc. Perkin Trans. 1, 621-, 1996; (b) Evans et al,J. Chem. Soc. Perkin Trans. 1, 103, 2127, 1981; (c) Oikawa et al, Tet.Lett, 37, 6169, 1996). Carboxyl activation of alkylsuccinate ester (24)followed by addition of morpholine in dimethylformamide and subsequentester hydroylsis, provides2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid (25). Couplingof (25) to the solid phase construct (11) followed by cleavage, provides(26), an example of general formula (I) where R=‘H’, (X)_(o)=‘CH₂’ i.e.R⁷, R⁸=‘H’, o=1, (W)_(n)=‘CO’, n=1, (V)_(m)=‘-’ and U=morpholino. Tothose skilled in the practices of organic synthesis, a wide variety ofalkylsuccinate esters such as (24) may be prepared and converted to thecorresponding substituted alkylsuccinate acid such as (25) following thegeneral conditions detailed. Additionally, morpholine may be replaced byany reasonable amine, providing many variations of carboxylic acid (25)following the general conditions detailed. Thus analogues of (26)exploring a wide range of (X)_(o), (V)_(m) and U in general formula (I)may be prepared through the general conditions detailed in Scheme 7.

Alternatively, (Scheme 8), a wide range of biarylalkylacetic acids,exemplified by 2RS-biphenyl-3-yl-4-methylpentanoic acid (28) are readilyavailable by known methods (see (a) DesJarlais, R. L. et al, J. Am.Chem. Soc, 120, 9114-9115, 1998; (b) Oballa, R. M. et al, WO 0149288).Coupling of biarylalkylacetic acid (28) to the solid phase construct(11) followed by cleavage, provides (29), an example of general formula(I) where R⁵=‘H’, (X)_(o)=‘-’, (W)_(n)=‘-’, (V)_(m)=‘-’ andU=m-biphenyl. To those skilled in the practices of organic synthesis, awide variety of biarylalkylacetic acids such as (28) may be prepared byalkylation of the α-anion of the free acid analogue of (27), which inturn is prepared by Suzuki coupling of phenylboronic acid and3-bromophenylacetic acid methyl ester. Phenylboronic acid may bereplaced by a wide range of arylboronic acids in the Suzuki coupling,providing many variations of carboxylic acid (28) following the generalconditions detailed. Thus analogues of (29) exploring a wide range ofgroup ‘U’ in general formula (I) may be prepared through the generalconditions detailed in Scheme 8.

Many other possibilities for solid phase organic chemistry (e.g. seeBrown, R. D. J. Chem. Soc., Perkin Trans.1, 19, 3293-3320, 1998, for areview of recent SPOC publications) can be used to derivatise construct(11) towards compounds of general formula (I). For example, theleft-hand portion ‘U—V—W—X—Y’ in general formulae (I) can be partiallyconstructed in solution, coupled to construct (11) and further modifiedon the solid phase. For example (Scheme 9), a simple extension of Scheme5 is through the oxidation of the intermediate solid phase boundspecies, with m-chloroperbenzoic acid in dichloromethane prior tocleavage, to give the sulphone analogue (30), an example of generalformula (I) where R⁵=‘H’, (X)_(o)=‘-’, (W)_(n)=‘SO₂’, n=1,(V)_(m)=‘CH₂’, i.e. R¹⁰, R¹¹=‘H’, m=1 and U=4-tert-butylphenyl. Asdescribed in Scheme 5, many variations of carboxylic acid (19) may beprepared following the general conditions detailed. Thus analogues of(30) exploring a wide range of (V)_(m) and U in general formula (I) maybe prepared through the general conditions detailed in Schemes 5 and 9.

Compounds of general formula (I) are finally released from the solidphase by treatment with trifluoroacetic acid/water, followed byevaporation, lyophylis and standard analytical characterisation.

A second strategy for the synthesis of compounds of general formula (I)comprises:

-   (f) Preparation of an appropriately functionalised and protected    (3aR, 6aR) N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide,    (3aS,6aR) N-(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide,    (3aR,6aS) N-(3-oxo-hexahydropentalen-3a-yl)alkylamide or (3aR,6aR)    N-(3-oxo-hexahydrocyclo penta[b]pyrrol-3a-yl)alkylamide building    block in solution. Preferred protecting groups for solution phase    chemistry are the Nα-tert-butoxycarbonyl group and the    Nα-benzyloxycarbonyl group.-   (g) Standard organic chemistry methods for the conversion of    building block (f) towards compounds of general formula (I), (Scheme    10).

An attractive alternative to the mixed anhydride activation of (31) isthrough the use of the pre-formed acyl fluoride (akin to that detailedin Scheme 1). The general strategy detailed in Scheme 10 is particularlyuseful when the compound of general formula (I) contains a substituentthat is labile to trifluoroacetic acid, this being the final reagentused in each of the solid phase Schemes 4-9. For example (Scheme 11),treatment in solution of α-hydroxyacid (35) with sodium hydride in adimethylformamide/dichloromethane mixture followed by addition of4-tert-butylbenzyl bromide, provides2RS-(4-tert-butylbenzyloxy)-4-methylpentanoic acid (36). Coupling of(36) to hydrochloride salt (34), provides (37), an example of generalformula (I) where R⁵=‘H’, (X)_(o)=‘-’, (W)_(n)=‘O’, n=1, (V)_(m)=‘CH₂’,i.e. R¹⁰, R¹¹=‘H’, m=1 and U=4-tert-butylphenyl. To those skilled in thepractices of organic synthesis, 4-tert-butylbenzyl bromide may bereplaced by any reasonable Ar—CR¹⁰R¹¹-halide, providing many variationsof carboxylic acid (36) under the conditions shown. Thus analogues of(37) exploring a wide range of (V)_(m) and U in general formula (I) maybe prepared through the conditions detailed in Scheme 11.

A third strategy for the synthesis of compounds of general formula (I)where the addition of U—V—W—X—Y to the protected (3aR,6aR)N-(3-oxo-hexahydrocyclo-penta[b]furan-3a-yl)alkylamide, (3aS,6aR)N-(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide, (3aR,6aS)N-(3-oxo-hexahydropentalen-3a-yl)alkyl amide or (3aR,6aR)N-(3-oxo-hexahydrocyclopenta[b]pyrrol-3a-yl)alkylamide building blockinvolves multistep organic reactions comprises:

-   (h) Preparation of an appropriately functionalised and protected    (3aR, 6aR) N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkylamide,    (3aS, 6aR) N-(3-oxo-hexa    hydrocyclopenta[b]thiophen-3a-yl)alkylamide, (3aR, 6aS)    N-(3-oxo-hexahydro pentalen-3a-yl)alkylamide or (3aR,6aR)    N-(3-oxo-hexahydrocyclopenta [b]pyrrol-3a-yl)alkylamide building    block in solution. Preferred protecting groups for solution phase    chemistry are the Nα-tert-butoxycarbonyl group and the    Nα-benzyloxycarbonyl group.-   (i) Protection of the ketone functionality of the (3aR, 6aR)    N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)alkyl amide, (3aS, 6aR)    N-(3-oxo-hexahydrocyclopenta[b]thiophen-3a-yl)alkylamide, (3aR, 6aS)    N-(3-oxo-hexahydropentalen-3a-yl)alkylamide or (3aR, 6aR)    N-(3-oxo-hexahydrocyclo penta[b]pyrrol-3a-yl)alkylamide building    block e.g. as a dimethylacetal.

Alternatively, the ketone may be reduced to the achiral secondaryalcohols and re-oxidised as the final synthetic step.

-   (j) Standard organic chemistry methods for the conversion of    building block (i) towards compounds of general formula (I).

Intermediates may be prepared in solution, followed by coupling tobuilding block (i) and further derivitisation towards compounds ofgeneral formula (I) (see Scheme 12 exemplified by preparation and use ofthe (3-hydroxyhexahydrocyclopenta[b]furan-3a-yl)carbamic acid tert-butylester (38)).

Alternatively, depending upon the types of chemistry used to constructthe left hand side U—V—W—X—Y of compounds of general formula (I), theketone may require protection e.g. as the dimethyl acetal. Such a methodis detailed and exemplified in Scheme 13 by the preparation and use of(3,3-dimethoxyhexahydrocyclo penta[b]furan-3a-yl)carbamic acid benzylester (42).

The invention extends to novel intermediates as described above, and toprocesses for preparing compounds of general formula (I) from each ofits immediate precursors. In turn, processes for preparing intermediatesfrom their immediate precursors also form part of the invention.

Compounds of general formula (I) are useful both as laboratory tools andas therapeutic agents. In the laboratory certain compounds of theinvention are useful in establishing whether a known or newly discoveredcysteine protease contributes a critical or at least significantbiochemical function during the establishment or progression of adisease state, a process commonly referred to as ‘target validation’.

According to a second aspect of the invention, there is provided amethod of validating a known or putative cysteine protease as atherapeutic target, the method comprising:

(a) assessing the in vitro binding of a compound as described above toan isolated known or putative cysteine protease, providing a measure ofpotency; and optionally, one or more of the steps of:

(b) assessing the binding of the compound to closely related homologousproteases of the target and general housekeeping proteases (e.g.trypsin) to provides a measure of selectivity;

(c) monitoring a cell-based functional marker of a particular cysteineprotease activity, in the presence of the compound; and

(d) monitoring an animal model-based functional marker of a particularcysteine protease activity in the presence of the compound.

The invention therefore provides a method of validating a known orputative cysteine protease as a therapeutic target. Differing approachesand levels of complexity are appropriate to the effective inhibition and‘validation’ of a particular target. In the first instance, the methodcomprises assessing the in vitro binding of a compound of generalformula (I) to an isolated known or putative cysteine protease,providing a measure of ‘potency’. An additional assessment of thebinding of a compound of general formula (I) to closely relatedhomologous proteases of the target and general house-keeping proteases(e.g. trypsin) provides a measure of ‘selectivity’. A second level ofcomplexity may be assessed by monitoring a cell-based functional markerof a particular cysteine protease activity, in the presence of acompound of general formula (I). For example, a ‘human osteoclastresorption assay’ has been utilised as a cell-based secondary in vitrotesting system for monitoring the activity of cathepsin K and thebiochemical effect of protease inhibitors (e.g. see WO-A-9850533). An‘MHC-II processing—T-cell activation assay’ has been utilised as acell-based secondary in vitro testing system for monitoring the activityof cathepsin S and the biochemical effect of protease inhibitors (Shi.G-P., et al, Immunity, 10, 197-206, 1999). When investigating viral orbacterial infections such a marker could simply be a functionalassessment of viral (e.g. count of mRNA copies) or bacterial loading andassessing the biochemical effect of protease inhibitors. A third levelof complexity may be assessed by monitoring an animal model-basedfunctional marker of a particular cysteine protease activity, in thepresence of a compound of general formula (I). For example, murinemodels of Leishmania infection, P. vinckei infection, malaria(inhibition of falcipain) and T. cruzi infection (cruzipain), indicatethat inhibition of cysteine proteases that play a key role in pathogenpropagation is effective in arresting disease symptoms, ‘validating’said targets.

The invention therefore extends to the use of a compound of generalformula (I) in the validation of a known or putative cysteine proteaseas a therapeutic target.

Compounds of general formula (I) are useful for the in vivo treatment orprevention of diseases in which participation of a cysteine protease isimplicated.

According to a third aspect of the invention, there is provided acompound of general formula (I) for use in medicine, especially forpreventing or treating diseases in which the disease pathology may bemodified by inhibiting a cysteine protease.

According to a fourth aspect of the invention, there is provided the useof a compound of general formula (I) in the preparation of a medicamentfor preventing or treating diseases in which the disease pathology maybe modified by inhibiting a cysteine protease.

Certain cysteine proteases function in the normal physiological processof protein degradation in animals, including humans, e.g. in thedegradation of connective tissue. However, elevated levels of theseenzymes in the body can result in pathological conditions leading todisease. Thus, cysteine proteases have been implicated in variousdisease states, including but not limited to, infections by Pneumocystiscarinii, Trypsanoma cruzi, Trypsanoma brucei brucei and Crithidiafusiculata; as well as in osteoporosis, autoimmunity, schistosomiasis,malaria, tumour metastasis, metachromatic leukodystrophy, musculardystrophy, amytrophy, and the like. See WO-A-9404172 and EP-A-0603873and references cited in both of them. Additionally, a secreted bacterialcysteine protease from S. Aureus called staphylopain has been implicatedas a bacterial virulence factor (Potempa, J., et al. J. Biol. Chem,262(6), 2664-2667, 1998).

The invention is useful in the prevention and/or treatment of each ofthe disease states mentioned or implied above. The present inventionalso is useful in a methods of treatment or prevention of diseasescaused by pathological levels of cysteine proteases, particularlycysteine proteases of the papain superfamily, which methods compriseadministering to an animal, particularly a mammal, most particularly ahuman, in need thereof a compound of the present invention. The presentinvention particularly provides methods for treating diseases in whichcysteine proteases are implicated, including infections by Pneumocystiscarinii, Trypsanoma cruzi, Trypsanoma brucei, Leishmania mexicana,Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth diseasevirus and Crithidia fusiculata; as well as in osteoporosis,autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromaticleukodystrophy, muscular dystrophy and amytrophy.

Inhibitors of cruzipain, particularly cruzipain-specific compounds, areuseful for the treatment of Chagas' disease.

In accordance with this invention, an effective amount of a compound ofgeneral formula (I) may be administered to inhibit the proteaseimplicated with a particular condition or disease. Of course, thisdosage amount will further be modified according to the type ofadministration of the compound. For example, to achieve an “effectiveamount” for acute therapy, parenteral administration of a compound ofgeneral formula a) is preferred. An intravenous infusion of the compoundin 5% dextrose in water or normal saline, or a similar formulation withsuitable excipients, is most effective, although an intramuscular bolusinjection is also useful. Typically, the parenteral dose will be about0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in amanner to maintain the concentration of drug in the plasma at aconcentration effective to inhibit a cysteine protease. The compoundsmay be administered one to four times daily at a level to achieve atotal daily dose of about 0.4 to about 400 mg/kg/day. The precise amountof an inventive compound which is therapeutically effective, and theroute by which such compound is best administered, is readily determinedby one of ordinary skill in the art by comparing the blood level of theagent to the concentration required to have a therapeutic effect.Prodrugs of compounds of the present invention may be prepared by anysuitable method. For those compounds in which the prodrug moiety is aketone functionality, specifically ketals and/or hemiacetals, theconversion may be effected in accordance with conventional methods.

The compounds of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit bone resorption or to achieve any other therapeuticindication as disclosed herein. Typically, a pharmaceutical compositioncontaining the compound is administered at an oral dose of between about0.1 to about 50 mg/kg in a manner consistent with the condition of thepatient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention. The compounds of this invention, which may have goodbioavailability, may be tested in one of several biological assays todetermine the concentration of a compound which is required to have agiven pharmacological effect.

According to a fifth aspect of the invention, there is provided apharmaceutical or veterinary composition comprising one or morecompounds of general formula (I) and a pharmaceutically or veterinarilyacceptable carrier. Other active materials may also be present, as maybe considered appropriate or advisable for the disease or conditionbeing treated or prevented.

The carrier, or, if more than one be present, each of the carriers, mustbe acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the abovedefined active agent with the carrier. In general, the formulations areprepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. The invention extendsto methods for preparing a pharmaceutical composition comprisingbringing a compound of general formula (I) in conjunction or associationwith a pharmaceutically or veterinarily acceptable carrier or vehicle.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art. A tablet may be made by compression or moulding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the active agent ina free flowing form such as a powder or granules, optionally mixed witha binder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be optionally be coated or scored andmay be formulated so as to provide slow or controlled release of theactive agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Parenteral formulations will generally be sterile.

According to a sixth aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

Preferred features for each aspect of the invention are as for eachother aspect mutatis mutandis.

The invention will now be illustrated with the following examples:

Solution Phase Chemistry—General Methods

All solvents were purchased from ROMIL Ltd (Waterbeach, Cambridge, UK)at SpS or Hi-Dry grade unless otherwise stated. General peptidesynthesis reagents were obtained from Chem-Impex Intl. Inc. (Wood DaleIll. 60191. USA). Thin layer chromatography (TLC) was performed onpre-coated plates (Merck aluminium sheets silica 60 F254, part no.5554). Visualisation of compounds was achieved under ultraviolet light(254 nm) or by using an appropriate staining reagent. Flash columnpurification was performed on silica gel 60 (Merck 9385). All analyticalHPLC were obtained on Phenomenex Jupiter C₄, 5μ, 300 A, 250×4.6 mm,using mixtures of solvent A=0.1% aq trifluoroacetic acid (TFA) andsolvent B=90% acetonitrile/10% solvent A on automated Agilent systemswith 215 and/or 254 nm UV detection. Unless otherwise stated a gradientof 10-90% B in A over 25 minutes at 1.5 mL/min was performed for fullanalytical HPLC analysis. HPLC-MS analysis was performed on an Agilent1100 series LC/MSD, using automated Agilent HPLC systems, with agradient of 10-90% B in A over 10 minutes on Phenomenex Columbus C₈, 5μ,300 A, 50×2.0 mm at 0.4 mL/min. Nuclear magnetic resonance (NMR) wereobtained on a Bruker DPX400 (400 MHz 1H frequency; QXI probe) in thesolvents and temperature indicated. Chemical shifts are expressed inparts per million (δ) and are referenced to residual signals of thesolvent. Coupling constants (J) are expressed in Hz.

Solid Phase Chemistry—General Methods

Example inhibitors (1-12) were prepared through a combination ofsolution and solid phase Fmoc-based chemistries (see ‘Solid PhasePeptide Synthesis’, Atherton, E. and Sheppard, R. C., IRL Press Ltd,Oxford, UK, 1989, for a general description). An appropriately protectedand functionalised building block was prepared in solution (e.g.compound (8), Scheme 1), then reversibly attached to the solid phasethrough an appropriate linker. Rounds of coupling/deprotection/chemicalmodification e.g. oxidation were then performed until the full lengthdesired molecule was complete (Scheme 2). Example inhibitors (1-12) werethen released (cleaved) from the solid phase, analysed, purified andassayed for inhibition verses a range of proteases.

Generally, multipins (polyamide 1.2→10 μmole loadings, seewww.mimotopes.com) were used for the solid phase synthesis, although anysuitable solid phase surface could be chosen. In general, the 1.2 μmolegears were used to provide small scale crude examples for preliminaryscreening, whilst the 10 μmole crowns were used for scale-up synthesisand purification of preferred examples. Standard coupling and Fmocdeprotection methods were employed (see Grabowska, U. et al, J. Comb.Chem. 2(5), 475-490, 2000, for a thorough description of solid phasemultipin methodologies).

Preparation of Initial Assembly

Building Block-linker constructs (e.g.(10)) were carboxyl activated with2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 1 mole equivalent), 1-hydroxybenzotriazole.hydrate(HOBT, 1 mole equivalent) and N-methylmorpholine (NMM, 2 moleequivalents) in dimethylformamide (DMF, typically 1 to 10 mL) for 5minutes. Amino functionalised DA/MDA crowns or HEMA gears (10 μmole percrown/1.2 μmole per gear, 0.33 mole equivalent of total surface aminofunctionalisation compared to activated construct) were added, followedby additional DMF to cover the solid phase surface. The loading reactionwas left overnight. Following overnight loading, crowns/gears were takenthrough standard cycles washing, Fmoc deprotection and loadingquantification (see Grabowska, U. et al) to provide loaded BuildingBlock-linker constructs (e.g.(11)). Analysis indicated virtuallyquantitative loading in all examples.

Coupling Cycles

The coupling of standard Fmoc-aminoacids (10 or 20 mole equivalent) wereperformed via carboxyl activated with2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 10 or 20 mole equivalent),1-hydroxybenzotriazole.hydrate (HOBT, 10 or 20 mole equivalent) andN-methylmorpholine (N, 20 or 40 mole equivalents) in dimethylformamide,with pre-activation for 5 minutes. Activated species were dispensed tothe appropriate wells of a polypropylene 96-well plate (Beckman, 1 mLwells, 500 μL solution per well for crowns or 250 μL solution per wellfor gears) in a pattern required for synthesis. Loaded free aminoBuilding Block-linker constructs (e.g.(11)) were added and the couplingreaction left overnight. Following overnight coupling, crowns/gears weretaken through standard cycles washing and Fmoc deprotection (seeGrabowska, U. et al). Identical activation and coupling conditions wereused for the coupling of a range of carboxylic acids (R—COOH).Alternatively, chloroformates e.g. morpholine-4-carbonylchloride (10mole equivalent), were coupled in DMF with the addition of NMM (10 moleequivalents).

Acidolytic Cleavage Cycle

A mixture of 95% TFA/5% water was pre-dispensed into two polystyrene96-well plates (Beckman, 1 mL wells, 600 μL solution per well for crownsor 300 μL solution per well for gears) in a pattern corresponding tothat of the synthesis. The completed multipin assembly was added to thefirst plate (mother plate), the block covered in tin foil and cleavedfor 24 hours. The cleaved multipin assembly was then removed from thefirst plate and added to the second plate (washing plate) for 15minutes. The spent multipin assembly was then discarded and themother/washing plates evaporated on an HT-4 GeneVac plate evaporator.

ANALYSIS AND PURIFICATION OF CLEAVED EXAMPLES

-   (a) Ex 1.21 μmole Gears. 100 μL dimethylsulphoxide (DMSO) was added    to each post cleaved and dried washing plate well, thoroughly mixed,    transferred to the corresponding post cleaved and dried mother plate    well and again thoroughly mixed. 10 μL of this DMSO solution was    diluted to 100 μL with a 90% acetonitrile/10% 0.1% aq TFA mixture.    20 μL aliquots were analysed by HPLC-MS and full analytical HPLC. In    each case the crude example molecules gave the expected [M+H]⁺ ion    and an HPLC peak at >80% (by 215 nm UV analysis). This provided an    approximately 10 mM DMSO stock solution of good quality crude    examples for preliminary protease inhibitory screening.-   (b) Ex 10 μmole Crowns. 500 μL of a 90% acetonitrile/10% 0.1% aq TFA    mixture was added to each washing plate well, thoroughly mixed,    transferred to the corresponding mother plate well and again    thoroughly mixed. 5 μL of this solution was diluted to 100 μL with a    90% acetonitrile/10% 0.1% aq TFA mixture. 20 μL aliquots were    analysed by HPLC-MS and full analytical HPLC. In each case the crude    example molecules gave the expected [M+H]⁺ ion and an HPLC peak    at >80% (by 215 nm UV analysis). The polystyrene blocks containing    crude examples were then lyophilized.-   (c) Individual examples (ex (b)) were re-dissolved in a 1:1 mixture    of 0.1% aq TFA/acetonitrile (1 mL) and purified by semi-preparative    BHLC (Phenomenex Jupiter C₄, 5μ, 300 A, 250×10 mm, a 25-90% B in A    gradient over 25 mins, 4.0 mL/min, 215 nm UV detection). Fractions    were lyophilised into pre-tarred glass sample vials to provide    purified examples (typically 2 to 4 mg, 40 to 80% yield).-   (d) Purified examples were dissolved in an appropriate volume of    DMSO to provide a 10 mM stock solution, for accurate protease    inhibitory screening.

Example 1 (3aR, 6aR)4-tert-Butyl-N-[2-(4-hydroxyphenyl)-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]benzamide

Following the general details from Scheme 1, the required bicyclebuilding block (3aR, 6aR) (3-Oxo-hexahydrocyclopenta[b]furan-3a-yl)carbamic acid 9H-fluoren-9-ylmethyl ester (8) was prepared as follows.

(1) Preparation of (2S)-2-tert-Butoxycarbonylamino-3-phenylpropionicacid 2-oxo-cyclopentyl ester

a) A solution of cyclopentanone (11.6 ml, 130 mmol) in methanol (250 ml)was added drop-wise at 0° C. over 20 minutes to a stirred solution ofpotassium hydroxide (85% tech., 22.1 g, 335 mmol) in methanol (75 ml).The mixture was stirred at 0° C. for 30 minutes then 2-iodosylbenzoicacid (36.45 g, 138 mmol) was added in portions over 1 hour. The mixturewas allowed to warm to ambient temperature over 4 hours, then stirred atambient temperature for 20 hours. The majority of solvent was removed invacuo then the product was extracted into dichloromethane (400 ml), thenthe extracts were washed with water (2×250 ml), dried (Na₂SO₄) and thesolvent removed in vacuo to leave 2,2-dimethoxycyclopentanol as acolourless oil (11.98 g) which was used without further purification.

b) 4-(Dimethylamino)pyridine (1.0 g, 8.2 mmol) was added at 0° C. to astirred suspension of 2,2-dimethoxycyclopentanol (11.98 g, 82 mmol),(S)-2-tert-butyloxycarbonylamino-3-phenylpropionic acid (23.9 g, 90.3mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(23.6 g, 123.1 mmol) in dichloromethane (500 ml). The mixture wasstirred at 0° C. for 4 hours, then it was washed with water (2×300 ml)and saturated aqueous sodium chloride solution (200 ml), dried (Na₂SO₄)and the solvent removed in vacuo to leave(2S)-2-tert-butoxycarbonylamino-3-phenylpropionic acid 2,2-dimethoxycyclopentyl ester as yellow oil (36.0 g) which was used without furtherpurification.

c) 4-Toluenesulphonic acid monohydrate (1.7 g, 9.2 mmol) was added to astirred solution of (2S)-2-tert-butoxycarbonylamino-3-phenylpropionicacid 2,2-dimethoxycyclopentyl ester (36 g, 91.6 mmol) in acetone (450ml) at ambient temperature. The solution was stirred for 3 days thenwater (600 ml) and saturated aqueous sodium hydrogen carbonate solution(200 ml) were added, then the product was extracted into ethyl acetate(600 ml). The aqueous phase was extracted with ethyl acetate (2×400 ml),then the combined ethyl acetate solutions were washed with saturatedaqueous sodium chloride solution (2×150 ml), dried (Na₂SO₄) and thesolvent removed in vacua. The residue (18.05 g) was purified by flashchromatography over silica gel eluting with a gradient of heptane:ethylacetate 3:1→2:1. Appropriate fractions were combined and the solventsremoved in vacuo to leave(2S)-2-tert-butoxycarbonylamino-3-phenylpropionic acid 2-oxocyclopentylester as a colourless oil (18.05 g, 40% from cyclopentanone). TLC(single UV spot, Rf=0.25, heptane:ethyl acetate 7:3), analytical HPLCwith main broad peak Rt=17.9-19.2 mins, HPLC-MS (main broad UV peak withRt=9.04-9.24 mins, 248.1 [M−Boc+2H]⁺, 370.2 [M+Na]⁺, 717.3 [2M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.42 (9H, 3×CH ₃, s), 1.79-2.48 (6H,3×cyclopentyl CH ₂, m), 3.06-3.28 (2H, CH ₂Ph, m), 4.60-5.20 (3H,COCHO+CHN+NH, m), 7.17-7.36 (5H, aromatic).(2) Preparation of (4aS, 7aS)3S-Benzyl-2-oxo-hexahydrocyclopenta[1,4]oxazine-4aS-carbonitrile

Trifluoroacetic acid (75 ml) was added drop-wise at 0° C. over 60minutes to a stirred solution of(2S)-2-tert-butoxycarbonylamino-3-phenylpropionic acid 2-oxocyclopentylester (17.05 g, 49.1 mmol) in dichloromethane (250 ml). The mixture wasstirred at 0° C. for 75 minutes then the majority of solvent was removedin vacuo. Toluene (75 ml) was added to the residue then the solvent wasremoved in vacuo to obtain an oil which was dissolved in acetonitrile(700 ml). Magnesium sulphate (29.5 g) then sodium acetate (20.1 g) wereadded to the stirred solution. The resulting suspension was stirred for90 minutes then solids were removed by filtration, then solvents removedin vacuo. The residue was dissolved in propan-2-ol (650 ml) then stirredunder nitrogen. Trimethylsilyl cyanide (13.1 ml, 98.4 mmol) was addeddrop-wise over 15 minutes then zinc chloride (49 ml, 1M solution indiethyl ether) was added over 40 minutes. The mixture was stirred for 18hours then cautiously added to saturated aqueous sodium hydrogencarbonate solution (750 ml). The mixture was diluted with water (750 ml)then the product was extracted into diethyl ether (3×500 ml). Thecombined ethereal solutions were washed with saturated aqueous sodiumchloride solution (350 ml), dried (MgSO₄) and the solvents removed invacuo to obtain a brown oil (10.05 g) which was purified by flashchromatography over silica gel eluting with a gradient of heptane:ethylacetate 4:1→3:2. Appropriate fractions were combined and the solventsremoved in vacuo to leave (3S, 4aR, 7aS)3-benzyl-2-oxohexahydrocyclopenta[1,4]oxazine-4a-carbonitrile as a whitesolid (4.54 g, 36%). TLC (single UV spot, Rf=0.45, heptane:ethyl acetate3:1), analytical HPLC Rt=14.521 mins, HPLC-MS (single main UV peak withRt=7.645 mins, 257.2 [M+H]⁺, 279 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.70-2.29 (7H, 3×CH ₂+NH, m), 2.84 (1H, CH ₂CHN,dd, J=14.3, 8.8 Hz), 3.52 (1H, CH ₂CHN, dd, J=14.3, 3.6 Hz), 3.90 (1H,CH₂CHN, dd, J=9.8, 3.6 Hz), 4.74 (1H, CHO, dd, J=6.8, 5 Hz), 7.15-7.32(5H aromatic).(3) Preparation of 1R-Amino-2S-hydroxycyclopentanecarboxylic acid

tert-Butylhypochlorite (4.0 ml, 35.4 mmol) was added drop-wise undernitrogen at 0° C. over 2 minutes to a stirred suspension of (3S, 4aR,7aS) 3-benzyl-2-oxohexahydrocyclopenta[1,4]oxazine-4a-carbonitrile (4.53g, 17.7 mmol) in diethyl ether (350 ml). The mixture was stirred at 0°C. for 140 minutes then triethylamine (7.4 ml, 53 mmol) was addeddrop-wise over 30 minutes. The resulting suspension was stirred at 0° C.for 3 hours then at ambient temperature for 23 hours. Insolublematerials were removed by filtration, then the filtrate was concentratedin vacuo. The residue was purified by flash chromatography over silicagel eluting with heptane:ethyl acetate (7:3). Appropriate fractions werecombined and the solvents removed in vacuo to leave a white solid (3.3g, TLC [single UV spot, Rf=0.3, heptane:ethyl acetate 2:1], analyticalHPLC Rt=16.071 mins) which was cooled to 0° C. then concentratedhydrochloric acid at 0° C. was added in one portion. The suspension wasallowed to warm to ambient temperature over 2 hours then stirred for 20hours. The reaction mixture was partitioned equally between six pressurevessels that were sealed then heated at 100° C. for 26 hours thenallowed to cool to ambient temperature. The reaction mixtures wererecombined then the product extracted into water (400 ml) then washedwith diethyl ether (2×200 ml) and the solvents removed in vacuo to leavea residue that was purified over Dowex 50WX4-200 ion exchange resineluting consecutively with 0.01M hydrochloric acid, water and then 1.0Maqueous ammonium hydroxide solution. Appropriate fractions were combinedand the solvents removed in vacuo to leave a solid which wasfreeze-dried from a mixture of water:acetonitrile (1:1) three times toobtain (1R, 2S) 1-amino-2-hydroxycyclopentanecarboxylic acid as a lightbrown solid (1.73 g, 67%). HPLC-MS (not UV active Rt=0.541 mins, 146.1[M+H]⁺).

_(δ)H (D₂O at 298K); 1.50-1.90 (4H, 2×CH₂, m), 2.16-2.25 (2H, CH₂, m),4.36 (1H, CHOH, dd, J=8.3, 7.7 Hz).

_(δ)C (D₂O at 298K); 21.56 (d, CH₂ CH₂CH₂), 33.46 and 35.00 (both d,CH₂CH₂ CH₂), 69.74 (CNH2), 77.54 (u, CHOH), 178.33 CO₂H).

(4) Preparation of1R-(9H-Fluoren-9-ylmethoxycarbonylamino)-2S-hydroxycyclopentanecarboxylic acid

(0.67 g, 4.6 mmol) was added at 0° C. to a stirred solution of sodiumcarbonate (1.0 g, 9.7 mmol) in water:1,4-dioxan (2:1, 45 ml). A solutionof 9-fluorenylmethyl chloroformate (1.25 g, 4.85 mmol) in 1,4-dioxan (15ml) was added drop-wise over 30 minutes. The resultant suspension wasstirred for 75 minutes at 0° C. then at ambient temperature for 45minutes. Water (200 ml) was added then the cloudy solution washed withchloroform (2×140 ml). Chloroform (100 ml) was added and the mixtureacidified with 1M hydrochloric acid (pH ˜2). The chloroform layer wasseparated then the aqueous layer re-extracted with chloroform (2×100ml). The chloroform extracts which had been separated from the acidifiedaqueous layer were combined then dried (Na₂SO₄) and the solvent removedin vacuo to leave a colourless oil to which heptane (100 ml) was addedbefore storing at −80° C. for 16 hours. The solvent was rapidly decantedfrom the oily residue which was washed with heptane (5 ml) thenremaining solvents removed in vacuo to obtain (1R, 2S)1-(9H-fluoren-9-ylmethoxycarbonylamino)-2-hydroxy cyclopentanecarboxylicacid as an oil (1.27 g, 75%). TLC (main UV spot, Rf=0.20, minor UV spot,Rf=0.15, 20% MeOH in CHCl₃), analytical HPLC Rt=17.172 mins (major),Rt=16.800 mins (minor) and HPLC-MS (main UV peak with Rt=7.840 mins,368.1 [M+H]⁺, 390.1 [M+Na]⁺, minor UV peak with Rt=7.646 mins, 368.1[M+H]⁺, 390.1 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.60-2.16 (4H, CH ₂CH ₂CH₂ and 1H, CH₂CH₂CH ₂,m), 2.35 (1H, CH₂CH₂CH ₂, m), 4.10 (1H, OH, brs), 4.24 (1H, Fmoc H-9,m), 4.36-4.57 (3H, Fmoc CH ₂ and CHOH, m), 5.93 (1H, NH, s), 7.28-7.33(2H aromatic, Fmoc H-2 and H-7), 7.34-7.41 (2H aromatic, Fmoc H-3 andH-6), 7.54-7.62 (2H aromatic, Fmoc H-1 and H-8), 7.72-7.79 (2H aromatic,Fmoc H-4 and H-5).(5) Preparation of1R-9H-Fluoren-9-ylmethoxycarbonylamino)-2S-hydroxycyclopentanecarboxylic acid allyl ester

A solution of 1R-(9H-fluoren-9-ylmethoxycarbonylamino)-2S-hydroxycyclopentanecarboxylic acid (1.75 g, 4.8 mmol) andtricaprylmethylammonium chloride (1.93 g, 4.8 mmol) in dichloromethane(14 ml) was added to a stirred solution of sodium hydrogen carbonate(0.4 g, 4.8 mmol) in water (14 ml), then allyl bromide (1.44 ml, 16.7mmol) was added in one portion. The biphasic mixture was stirred for 20hours then diluted with water (50 ml) and the product extracted intodichloromethane (2×50 ml). The combined organic layers were dried(MgSO₄) and the solvent removed in vacuo to obtain a colourless oilwhich was purified by flash chromatography over silica gel eluting witha gradient of heptane:ethyl acetate 10:3→20:7. Appropriate fractionswere combined and the solvents removed in vacuo to leave (1R, 2S)1-(9H-fluoren-9-ylmethoxycarbonylamino)-2-hydroxycyclopentanecarboxylicacid allyl ester as a colourless oil (1.32 g, 67%). TLC (single UV spot,Rf=0.25, heptane:ethyl acetate 3:1), analytical HPLC Rt=20.371 mins(major), Rt=19.706 min (minor) and HPLC-MS (main UV peak with Rt=9.412mins, 408.1 [M+H]⁺, 430.1 [M+Na]⁺; minor UV peak with Rt=9.102 mins,408.1 [M+H]⁺, 430.1 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.6-2.63 (7H, CH ₂CH ₂CH₂, OH, m), 4.8-4.27 (1H,Fmoc H-9, m), 4.29-4.48 (3H, H-2 and Fmoc CH ₂, m), 4.57-4.66 (2H, CH₂CH═CH₂, brs), 5.22 (1H, CH₂CH═CH ₂, dd, J=10.4, 1.0 Hz), 5.29 (1H,CH₂CH═CH ₂, d, J=13.6 Hz), 5.77 (1H, NH, brs), 5.82-5.94 (1H, CH₂CH═CH₂,m), 7.27-7.32 (2H aromatic, Fmoc H-2 and H-7), 7.36-7.41 (2H aromatic,Fmoc H-3 and H-6), 7.55-7.62 (2H aromatic, Fmoc H-1 and H-8), 7.74-7.77(2H aromatic, Fmoc H-4 and H-5).

_(δ)C (CDCl₃ at 298K); 20.37 (d, CH₂ CH₂CH₂), 32.52/32.29 and 34.20(both d, CH₂CH₂ CH₂), 47.58/47.52 (u, Fmoc C-9), 66.53 (d, Fmoc CH₂),67.34 (d, CH₂CH═CH₂), 76.0 (q, CCO₂CH₂), CHOH under CHCl₃?, 118.97 (d,CH₂═CHCH₂), 120.39 (u, Fmoc C-4 and C-5), 125.49 (u, Fmoc C-1 and C-8),127.45/127.46 (u, Fmoc C-2 and C-7), 128.09 (u, Fmoc C-3 and C-6),132.09 (u, CH₂═CHCH₂), 141.72 (q, Fmoc C-4′ and C-5′), 144.20/144.33 (q,Fmoc C-1′ and C-8′), 156.76 (q, OCON), 173.61 (q, CO₂CH₂CH═CH₂).(6) Preparation of 1R-(9H-Fluoren-9-ylmethoxycarbonylamino)-2-oxo-cyclopentanecarboxylic acid allyl ester

A solution of dimethyl sulphoxide (0.224 ml, 3.15 mmol) indichloromethane (1.0 ml) was added under nitrogen to a stirred solutionof oxalyl chloride (0.132 ml, 1.51 mmol) in dichloromethane (2.5 ml) at−70° C. over 20 minutes. The mixture was stirred for 10 minutes then asolution of (1R, 2S)1-(9H-fluoren-9-ylmethoxycarbonylamino)-2-hydroxycyclopentanecarboxylicacid allyl ester (0.535 g, 1.3 mmol) in dichloromethane (3 ml) addedover 20 minutes. The mixture was stirred for 10 minutes thentriethylamine (0.92 ml, 6.57 mmol) added drop-wise over 5 minutes. Thecooling bath was then removed and stirring continued for 45 minutes atambient temperature. Saturated aqueous ammonium chloride solution (50ml) was added then the product extracted into diethyl ether (2×50 ml).The combined ethereal layers were washed with water (25 ml), dried(MgSO₄) and the solvent was removed in vacuo to obtain a colourless oil(520 mg) which was purified by flash chromatography over silica geleluting with heptane:ethyl acetate (3:1). Appropriate fractions werecombined and the solvents removed in vacuo to leave (1R)1-(9H-fluoren-9-ylmethoxycarbonylamino)-2-oxocyclopentane carboxylicacid allyl ester as a colourless oil (0.43 g, 81%). TLC (single UV spot,Rf=0.30, heptane:ethyl acetate 2:1), analytical HPLC with main peakRt=19.993 mins, HPLC-MS (single UV peak with Rt=10.132 mins, 406.1[M+H]⁺, 428.1 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 2.15-2.23 (2H, CH₂CH ₂CH₂, m), 2.46-2.70 (4H, CH₂CH₂CH ₂, m), 4.21 (1H, Fmoc H-9, t, J=7.1 Hz), 4.35 (2H, Fmoc CH ₂, d,J=7.1), 4.66 (2H, CH ₂CH═CH₂, brs), 5.26-5.35 (2H, CH₂CH═CH ₂, m),5.80-5.91 (1H, CH₂CH═CH₂, m), 6.14 (1H, NH, brs), 7.27-7.35 (2Haromatic, Fmoc H-2 and H-7), 7.36-7.41 (2H aromatic, Fmoc H-3 and H-6),7.54-7.60 (2H aromatic, Fmoc H-1 and H-8), 7.74-7.77 (2H aromatic, FmocH-4 and H-5).

_(δ)C (CDCl₃ at 298K); 19.19 (d, CH₂ CH₂CH₂), 34.53 and 36.87 (both d,CH₂CH₂ CH₂), 47.42 (u, Fmoc C-9), 67.43 and 67.665 (both d, Fmoc CH₂ andCH₂CH═CH₂), 67.85 (q, CCO₂CH₂), 119.92 (d, CH₂CH═CH₂), 120.40 (u, FmocC-4 and C-5), 125.52 (u, Fmoc C-1 and C-8), 127.51 (u, Fmoc C-2 andC-7), 128.16 (u, Fmoc C-3 and C-6), 131.09 (u, CH₂═CHCH₂), 141.68 (q,Fmoc C-4′ and C-5′), 144.00/144.16 (q, Fmoc C-1′ and C-8′), 155.25 (q,OCON), 169.25 (q, CO₂CH₂CH═CH₂) 211.30 (q, COCH₂CH₂).(7) Preparation of 1R-(9H-Fluoren-9-ylmethoxycarbonylamino)-2R-hydroxycyclopentanecarboxylic acid allyl ester

Sodium borohydride (39 mg, 1.04 mmol) was added to a stirred solution of1R-(9H-fluoren-9-ylmethoxycarbonylamino)-2-oxocyclopentanecarboxylicacid allyl ester (0.42 g, 1.04 mmol) in methanol (6 ml) at 0° C. in oneportion. The mixture was stirred for 10 min then solvents removed invacua to leave a residue. Water (20 ml) and dichloromethane (20 ml) wereadded followed by 1M hydrochloric acid to acidify the mixture (pH ˜1.5).The dichloromethane layer was collected then the aqueous layer extractedwith dichloromethane (20 ml). The combined dichloromethane layers werewashed with aqueous saturated sodium chloride solution (20 ml). Theaqueous saturated sodium chloride solution was extracted withdichloromethane (10 ml) then the dichloromethane layers were combinedthen dried (MgSO₄) and the solvent removed in vacuo to obtain acolourless oil (420 mg) which was purified by flash chromatography oversilica gel eluting with a gradient of heptane:ethyl acetate 7:3→13:7.Appropriate fractions were combined and the solvents removed in vacuo toleave 1R-(9H-fluoren-9-ylmethoxycarbonylamino)-2R-hydroxycyclopentanecarboxylic acid allyl ester as a colourless oil (288 mg, 68%). TLC(single UV spot, Rf=0.25, heptane:ethyl acetate 3:1), analytical HPLCRt=19.680 mins (major), Rt=20.323 min (minor) and HPLC-MS (main UV peakwith Rt=9.076 mins, 408.1 [M+H]⁺, 430.0 [M+Na]⁺; minor UV peak withRt=9.451 mins, 408.1 [M+H]⁺, 430.0 [M+Na]⁺). 4-Toluenesulphonic acidmonohydrate (30 mg, 0.16 mmol) was added to a solution of the oil (230mg) in toluene (12 ml) then the mixture heated at 100° C. for 75minutes. Two further batches (170 mg and 35 mg) of the oil (preparedusing the same procedure as above) were similarly treated with4-toluenesulphonic acid monohydrate with appropriate scaling ofquantities, then the three toluene mixtures were combined and solventsremoved in vacuo to obtain a residue which was purified by flashchromatography over silica gel eluting with a gradient of heptane:ethylacetate 7:3→13:7. Appropriate fractions were combined and the solventsremoved in vacuo to leave (1R, 2R)1-(9H-fluoren-9-ylmethoxycarbonylamino)-2-hydroxycyclopentanecarboxylicacid allyl ester as a colourless oil (380 mg). TLC (single UV spot,Rf=0.25, heptane:ethyl acetate 3:1), analytical HPLC single UV peak withRt=18.141 mins and HPLC-MS (single UV peak with Rt=9.140 mins, 408.1[M+H]⁺, 430.1 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.72-2.44 (6H, CH ₂CH ₂CH₂, m), 4.12-4.19 (1H,H-2, m) 4.27 (1H, Fmoc H-9, t, J=6.5 Hz), 4.37-4.55 (3H, Fmoc CH ₂ andOH, m), 4.60-4.75 (2H, CH ₂CH═CH₂, brs), 5.25 (1H, CH₂CH═CH ₂, d, J=10.5Hz), 5.30-5.39 (2H, CH₂CH═CH ₂ and NH, m), 5.83-5.96 (1H, CH₂CH═CH₂, m),7.29-7.35 (2H aromatic, Fmoc H-2 and H-7), 7.39-7.44 (2H aromatic, FmocH-3 and H-6), 7.58-7.65 (2H aromatic, Fmoc H-1 and H-8), 7.77-7.80 (2Haromatic, Fmoc H-4 and H-5).

_(δ)C (CDCl₃ at 298K); 20.92 (d, CH₂ CH₂CH₂), 32.46 and 35.63 (both d,CH₂CH₂ CH₂), 47.52 (u, Fmoc C-9), 66.55 and 67.37 (both d, Fmoc CH₂ andCH₂CH═CH₂), 69.43 (q, CCO₂CH₂), 80.50 (u, CHOH), 118.98 (d, CH₂═CHCH₂),120.43 (u, Fmoc C-4 and C-5), 125.41 (u, Fmoc C-1 and C-8),127.47/127.49 (u, Fmoc C-2 and C-7), 128.14/128.16 (u, Fmoc C-3 andC-6), 132.08 (u, CH₂═CHCH₂), 141.73 (q, Fmoc C-4′ and C-5′),144.00/144.23 (q, Fmoc C-1′ and C-8′), 156.86 (q, OCON), 172.99 (q,CO₂CH₂CH═CH₂).(8) Preparation of2R-tert-Butoxy-1R-(9H-Fluoren-9-ylmethoxycarbonylamino)cyclopentanecarboxylic acid allyl ester

A stirred solution of1R-(9H-fluoren-9-ylmethoxycarbonylamino)-2R-hydroxycyclopentanecarboxylic acid allyl ester (360 mg, 0.88 mmol) indichloromethane (5 ml) was cooled in a sealed pressure vessel to −70° C.then isobutylene gas (˜3 ml) condensed into the solution. Concentratedsulphuric acid (25 μl) was added then the pressure vessel sealed. Themixture was stirred at ambient temperature for 20 hours then cooled to−70° C. N-Methylmorpholine (50 μl) was added then the unsealed pressurevessel allowed to warm to ambient temperature. The mixture was dilutedwith saturated aqueous sodium hydrogen carbonate solution (50 ml) andwater (25 ml) then the product extracted into dichloromethane (50 mlthen 2×25 ml). The combined dichloromethane layers were washed withsaturated aqueous sodium chloride solution (25 ml), dried (Na₂SO₄) andthe solvent removed in vacuo. The residue (370 mg) was purified by flashchromatography over silica gel eluting with a gradient of heptane:ethylacetate 4:1→7:3. Appropriate fractions were combined and the solventsremoved in vacuo to leave2R-tert-butoxy-1R-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopentanecarboxylicacid allyl ester as a colourless oil (305 mg, 75%). TLC (single UV spot,Rf=0.50, heptane:ethyl acetate 2:1), analytical HPLC Rt=22.623 mins andHPLC-MS (single UV peak with Rt=11.611 mins, 408.1 [M−^(t)Bu+2H]⁺, 486.1[M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.06 (9H, ^(t)Bu, s), 1.80-2.41 (6H, CH ₂CH ₂CH₂, m), 4.21 (1H, Fmoc H-9, t, J=6.8 Hz), 4.26-4.50 (3H, Fmoc CH ₂ andH-2, m), 4.58-4.74 (1H, CH ₂CH═CH₂, brs), 5.21 (1H, CH₂CH═CH ₂, d,J=10.4 Hz), 5.35 (1H, CH₂CH═CH ₂, d, J=17.2 Hz), 5.84-6.01 (2H,CH₂CH═CH₂ and NH, m), 7.26-7.30 (2H aromatic, Fmoc H-2 and H-7),7.37-7.39 (2H aromatic, Fmoc H-3 and H-6), 7.57-7.61 (2H aromatic, FmocH-1 and H-8), 7.70-7.77 (2H aromatic, Fmoc H-4 and H-5).

_(δ)C (CDCl₃ at 298K); 21.69/21.47 (d, CH₂ CH₂CH₂), 32.98 and 33.82(both d, CH₂CH₂ CH₂), 47.63 (u, Fmoc C-9), 66.54 and 66.89 (both d, FmocCH₂ and CH₂CH═CH₂), 70.07 and 74.03 (both q, CCO₂CH₂ and OCMe₃), 79.51(u, CHOCMe₃), 118.53 (d, CH₂═CHCH₂), 120.38 (u, Fmoc C-4 and C-5),125.46 (u, Fmoc C-1 and C-8), 127.46 (u, Fmoc C-2 and C-7), 128.07 (u,Fmoc C-3 and C-6), 132.40 (u, CH₂═CHCH₂), 141.70 (q, Fmoc C-4′ andC-5′), 144.32 (q, Fmoc C-1′ and C-8′), 155.13 (q, OCON), 173.00 (q,CO₂CH₂CH═CH₂).(9) Preparation of2R-tert-Butoxy-1R-(9H-Fluoren-9-ylmethoxycarbonylamino)cyclopentanecarboxylic acid

Tetrakistriphenylphosphine palladium(0) (15 mg, 0.013 mmol),dichloromethane (5 ml) then phenyltrihydrosilane (153 μl, 1.24 mmol)were added consecutively to 2R-tert-butoxy-1R-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopentane carboxylic acid allyl ester (288 mg, 0.62mmol) under nitrogen. The mixture was stirred for 45 minutes then 0.01Mhydrochloric acid (30 ml) added and the product extracted intochloroform (1×20 ml then 1×10 ml). The combined chloroform layers weredried (Na₂SO₄) and the solvent removed in vacuo. The residue (460 mg)was purified by flash chromatography over silica gel eluting with agradient of heptane:ethyl acetate 2:1→1:3. Appropriate fractions werecombined and the solvents removed in vacuo to leave2R-tert-butoxy-1R-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopentanecarboxylicacid as a colourless oil (205 mg, 78%). TLC (single UV spot, Rf=0.25,heptane:ethyl acetate 1:2), analytical HPLC Rt=19.539 mins and HPLC-MS(single UV peak with Rt=9.850 mins, 368.1 [M−^(t)Bu+2H]⁺, 446.1[M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.22 (9H, ^(t)Bu, s), 1.77-2.37 (6H, CH ₂CH ₂CH₂, m), 4.22 (1H, Fmoc H-9, t, J=6.8 Hz), 4.27-4.36 (2H, Fmoc CH ₂, m),4.65 (1H, H-2, brs), 5.39 (1H, NH, brs), 7.27-7.35 (2H aromatic, FmocH-2 and H-7), 7.36-7.41 (2H aromatic, Fmoc H-3 and H-6), 7.56-7.61 (2Haromatic, Fmoc H-1 and H-8), 7.73-7.76 (2H aromatic, Fmoc H-4 and H-5).

_(δ)C (CDCl₃ at 298K); 21.48 (d, CH₂ CH₂CH₂), 33.70 and 35.31 (both d,CH₂CH₂ CH₂), 47.56 (u, Fmoc C-9), 67.22 (d, Fmoc CH₂), 68.77 (q, CCO₂CH₂or OCMe₃ other peak under chloroform?), 78.58 (u, CHOCMe₃), 120.37 (u,Fmoc C-4 and C-5), 125.54 (u, Fmoc C-1 and C-8), 127.47 (u, Fmoc C-2 andC-7), 128.09/128.12 (u, Fmoc C-3 and C-6), 141.69/141.73 (q, Fmoc C-4′and C-5′), 144.02/144.38 (q, Fmoc C-1′ and C-8′), 156.05 (q, OCON),174.89 (q, CO₂H).(10) Preparation of(2R-tert-Butoxy-1R-fluorocarbonyl-cyclopentyl)carbamic acid9H-fluoren-9-ylmethyl ester

Pyridine (53 μl, 0.66 mmol) then cyanuric fluoride (71 μl, 0.85 mmol)were added consecutively at 0° C. to a stirred solution of2R-tert-butoxy-1R-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopentanecarboxylicacid (159 mg, 0.38 mmol) in dichloromethane (5 ml) under nitrogen. Thesuspension was stirred for 30 minutes at 0° C. then for 5 hours atambient temperature. Crushed ice (˜10 ml) and ice-chilled water (10 ml)was added, then the product was extracted into dichloromethane (20 ml).The dichloromethane layer was dried (MgSO₄) and the solvent removed invacuo to give (2R-tert-butoxy-1R-fluorocarbonylcyclopentyl)carbamic acid9H-fluoren-9-ylmethyl ester as a pale brown oil (115 mg, 71%) which wasused without further purification. TLC (single UV spot, Rf=0.45,heptane:ethyl acetate 2:1), analytical HPLC main UV peak with Rt=23.933mins and HPLC-MS (main UV peak with Rt=11.439 mins, 370.1[M−^(t)Bu+2H]⁺, 448.1 [M+Na]⁺).(11) Preparation of (3aR, 6aR)(3-Oxo-hexahydrocyclopenta[b]furan-3a-yl)carbamic acid9H-fluoren-9-ylmethyl ester

a) Ethereal diazomethane [generated from diazald (0.94 g, ˜3 mmol)addition in diethyl ether (15 ml) to sodium hydroxide (1.05 g) in water(1.5 ml)/ethanol (3.0 ml) at 60° C.] was added to a stirred solution of(1R, 2R) (2-tert-butoxy-1-fluorocarbonylcyclopentyl)carbamic acid9H-fluoren-9-ylmethyl ester (115 mg, 0.27 mmol) in dichloromethane (2ml) at 0° C. The solution was stirred for 20 minutes at ° C. then atambient temperature for 20 hours. Acetic acid (0.6 ml, 10.5 mmol) wasadded then the solution was stirred for 5 minutes before addingtert-butyl methyl ether (50 ml). The ethereal layer was washed withsaturated aqueous sodium hydrogen carbonate solution (40 ml) then water(2×30 ml), then dried (Na₂SO₄) and the solvent removed in vacuo. Theresidue (130 mg) was purified by flash chromatography over silica geleluting with a gradient of heptane:ethyl acetate 4:1→1:3. Appropriatefractions were combined and the solvents removed in vacuo to leave[2R-tert-butoxy-1R-(2-diazoacetyl) cyclopentyl]carbamic acid9H-fluoren-9-ylmethyl ester (16 mg) as an oil which was used withoutfurther purification.

b) A solution of lithium chloride (15 mg, 0.36 mmol) in aceticacid:water (4:1, 1.0 ml) was added to[2R-tert-butoxy-1R-(2-diazoacetyl)cyclopentyl]carbamic acid9H-fluoren-9-ylmethyl ester (16 mg). The solution was stirred for 2.5hours then chloroform (25 ml) and saturated aqueous sodium hydrogencarbonate solution (25 ml) was added. The chloroform layer washed withsaturated aqueous sodium hydrogen carbonate solution (25 ml), saturatedaqueous sodium chloride solution (25 ml), dried (Na₂SO₄) and the solventremoved in vacuo. The residue (16 mg) was purified by flashchromatography over silica gel eluting with a gradient of heptane:ethylacetate 31:9→3:1. Appropriate fractions were combined and the solventsremoved in vacuo to leave (3aR, 6aR)(3-oxohexahydrocyclopenta[b]furan-3a-yl)carbamic acid9H-fluoren-9-ylmethyl ester (11.0 mg) as a white solid. TLC (single UVspot, Rf=0.3, heptane:ethyl acetate 2:1), analytical HPLC main UV peakwith Rt=18.872 mins and HPLC-MS (main UV peak with Rt=9.208 mins, 364.0[M+H]⁺, 386.0 [M+Na]⁺).

_(δ)H (CDCl₃ at 298K); 1.55-2.19 (6H, CH ₂CH ₂CH ₂, m), 4.15 (1H, H-2,d, J=16.8 Hz), 4.19 (1H, Fmoc H-9, t, J=6.7 Hz), 4.31 (1H, H-2, d,J=16.8 Hz), 4.36-4.44 (2H, Fmoc CH ₂, m), 4.74 and 4.97 (each 1H, H-6aand NH, brs), 7.29-7.36 (2H aromatic, Fmoc H-2 and H-7), 7.38-7.44 (2Haromatic, Fmoc H-3 and H-6), 7.53-7.61 (2H aromatic, Fmoc H-1 and H-8),7.74-7.80 (2H aromatic, Fmoc H-4 and H-5).

_(δ)C (CDCl₃ at 298K); 24 (CH₂ CH₂CH₂), 33 and 37 (CH₂CH₂ CH₂), 48 (u,Fmoc C-9), 68 (Fmoc CH₂), 70 (C-3a), 72 (d, C-2), 87 (u, C-6a), 120 (u,Fmoc C-4 and C-5), 125 (u, Fmoc C-1 and C-8), 127 (u, Fmoc C-2 and C-7),128 (u, Fmoc C-3 and C-6), 142 (Fmoc C-4′ and C-5′), 144 (Fmoc C-1′ andC-8′), 156 (OCON), 215 (C-3).

Following the general details from Scheme 2, the required bicyclebuilding block (3aR, 6aR) (3-Oxo-hexahydrocyclopenta[b]furan-3a-yl)carbamic acid 9H-fluoren-9-ylmethyl ester (8) was converted to buildingblock-linker construct (10) as follows:

(3aR, 6aR) (3-Oxo-hexahydrocyclopenta[b]furan-3a-yl) carbamic acid9H-fluoren-9-ylmethyl ester (8) (26.0 mg, 0.072 mmole) was dissolved ina mixture of ethanol (1.75 mL) and water (0.25 mL) containing sodiumacetate.trihydrate (14.6 mg, 0.107 mmole, 1.5 eq).4-[[(hydrazinocarbonyl)amino] methyl]cyclohexanecarboxylicacid.trifluoroacetate (23.6 mg, 0.072 mmole, 1.0 eq, Murphy, A. M. etal, J. Am. Chem. Soc., 114, 3156-3157, 1992) was added and the mixturerefluxed for 24 hr. Chloroform (35 mL) was added and the organics washedwith HCl (2×15 mL, ˜pH3), then brine (1×15 mL), dried (Na₂SO₄) andreduced in vacuo to provide crude building block-linker construct (10)as a colourless gum, yield 40.8 mg, analytical HPLC 2 product peaksRt=17.57 and 18.08 mins (cis/trans geometrical isomers), HPLC-MS (2×UVpeak, both with 561 [M+H]⁺, 1121 [2M+Na]⁺). Crude (10) was used directlyfor construct loading.

Following the general details from Scheme 2, the required buildingblock-linker construct (10) was attached to the solid phase providingloaded building block-linker construct (11) as follows:

Building block-linker construct (10) (0.066 mmole),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU, 25.0 mg, 0.066 mmole), 1-hydroxybenzotriazole.hydrate and (HOBT,10.1 mg, 0.066 mmole) were dissolved in dimethylformamide (2 mL) andN-methylmorpholine (NMM, 14.4 μL, 0.13 mmole) added. Afterpre-activation for 5 minutes, free amine gears (20×1.2 μmole) wereadded, followed by dimethylformamide (0.5 mL) and left overnight. Thespent coupling solution was then added to free amine crowns (2×10 μmole)and left overnight. Standard washing and analyses indicated loading at87%.

Following the general details from Scheme 2, the required loadedbuilding block-linker construct (11) was elaborated on the solid phaseas follows:

Loaded construct (11) was elaborated to EXAMPLE 1 (3aR, 6aR)4-tert-Butyl-N-[2-(4-hydroxyphenyl)-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]benzamide by standard Fmoc deprotection and sequential couplingwith Fmoc-Tyr(OBut)-OH then 4-tert-butylbenzoic acid. The crude examplewas cleaved and analysed (see general techniques). HPLC Rt=17.99 mins(>90%/), HPLC-MS 465.2 [M+H]⁺, 951.4 [2M+Na]⁺.

The following examples (2-12) were prepared as detailed for EXAMPLE 1,coupling with the required reagents to provide the full length molecule.

Example 2 (3aR, 6aR) Biphenyl-4-carboxylic acid[2-(4-hydroxyphenyl)-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]amide

HPLC Rt=17.43 mins (>95%), HPLC-MS 485.2 [M+H]⁺.

Example 3 (3aR, 6aR) Naphthalene-1-carboxylic acid[2-(4-hydroxyphenyl)-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]amide

HPLC Rt=15.195 mins (>95)%, HPLC-MS 459.2 [M+H]⁺.

Example 4 (3aR, 6aR) 4-tert-Butyl-N-[3-methyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl) butyl]benzamide

HPLC Rt=20.158 mins (>80%), HPLC-MS 415.1 [M+H]⁺, 851.3 [2M+Na]⁺.

Example 5 3aR, 6aR) Biphenyl-4-carboxylic acid-[3-methyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl) butyl]benzamide

HPLC Rt=19.53 mins (>85%), HPLC-MS 435.2 [M+H]⁺, 891.4 [2M+Na]⁺.

Example 6 (3aR, 6aR) Benzo[b]thiophene-2-carboxylic acid[3-methyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)butyl]amide

HPLC Rt=18.36 mins (>80%), HPLC-MS 415.1 [M+H]⁺, 851.3 [2M+Na]⁺.

Example 7 (3aR, 6aR) Thiophene-3-carboxylic acid[2-cyclohexyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)ethyl]amide

HPLC Rt=17.79 mins (>85%), HPLC-MS 405.2 [M+H]⁺, 831.3 [2M+Na]⁺.

Example 8 2RS, 3aR, 6aR) 2-Benzyloxy-3-cyclohexyl-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)propionamide

HPLC Rt=21.77-21.97 mins (>90%), HPLC-MS 386.3 [M+H]⁺

(a) Preparation of 3-Cyclohexyl-2S-hydroxypropionic acid (Compound (14)Scheme 4)

A solution of sodium nitrite (12.1 g, 175 mmol) in water (40 ml) wasadded dropwise to a stirred suspension of(S)-α-aminocyclohexanepropionic acid hydrate (5 g, 26.5 mmol) in 0.5Msulphuric acid (120 ml, 60 mmol) at 0° C. over 1.5 hours. The mixturewas allowed to warm to ambient temperature over 20 hours. The productwas extracted into diethyl ether (2×25 ml) then the ethereal layers werewashed with saturated aqueous sodium chloride solution (2×25 ml), dried(Na₂SO₄) and the solvents removed in vacuo. The residue (5.3 g) wasrecrystallized from diethyl ether (10 ml) and heptane (25 ml) to give3-cyclohexyl-2S-hydroxypropionic acid as a white solid, yield 2.4 g,(53%).

δ_(H) (400 MHz, CDCl₃ at 298K), 0.89-1.35 (5H, m) and 1.51-1.86 (7H, m)(OCHCH ₂ and cyclohexyl), 4.32 (1H, OCHCH₂, m)

(b) Preparation of 2RS-Benzyloxy-3-cyclohexylpropionic acid (Compound(15) Scheme 4)

Sodium hydride (265 mg of 60% dispersion in oil, 6.6 mmol) was added intwo portions to a stirred mixture of 3-cyclohexyl-2S-hydroxypropionicacid (0.52 g, 3.0 mmol), dimethylformamide (5 ml) and dichloromethane (5ml) at 0° C. over 5 minutes. The mixture was stirred at 0° C. for 5minutes then at ambient temperature for 45 minutes. Benzyl bromide (0.45ml, 3.8 mmol) was added then the mixture stirred for 1 hour beforeadding dimethylformamide (5 ml). After stirring for 4 hours potassiumiodide (50 mg, 0.3 mmol) was added. The mixture was stirred for 20 hoursthen heated at 55° C. for 1 hour then allowed to cool to ambienttemperature and poured into water (15 ml). A saturated aqueous sodiumchloride solution (5 ml) was added then the mixture was extracted withdichloromethane (5 ml then 10 ml) that was discarded. The aqueous layerwas acidified using 1M hydrochloric acid (10 ml) then extracted withdichloromethane (2×10 ml). The dichloromethane layer was dried (MgSO₄)and the solvent removed in vacuo. The residue (0.55 g) was dissolved indimethylformamide (8 ml) then cooled to 0° C. before adding sodiumhydride (190 mg of 60% dispersion in oil, 4.75 mmol). The mixture wasstirred for 30 minutes then polymer bound isocyanate (380 mg, 2mmolNg⁻¹) added. The mixture was stirred for 2 hours at ambienttemperature then poured into water (15 ml). 1M Hydrochloric acid (10 ml)was added then the product was extracted into dichloromethane (2×10 ml),dried (Na₂SO₄) and the solvent removed in vacuo. The residue waspurified by flash chromatography over silica gel eluting with a gradientof methanol:dichloromethane 0:1→1:20. Appropriate fractions werecombined and the solvents removed in vacuo to give2RS-benzyloxy-3-cyclohexylpropionic acid as a colourless oil, yield 41mg (5.2%).

HPLC-MS (single main UV peak with Rt=9.47 mins, 261.2 [M−H]⁻,285.2[M+Na]⁺, 547.3[2M+Na]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K), 0.72-1.03 (2H, cyclohexane, m),1.08-1.38 (3H, cyclohexane, m), 1.45-1.93 (6H+2Hβ, cyclohexane, m),3.93-4.18 (1Hα, OCHCO), 4.35-4.53 (1H, CH ₂O, d, J=11.52 Hz), 4.68-4.88(1H CH ₂O, d, J=11.54 Hz), 7.20-7.47 (5H, ArH, m), 9.36 (1H, OH, brs).

Compound (15) was coupled under standard conditions to loaded buildingblock-linker construct (11) (following standard removal of Fmoc), thencleaved to provide EXAMPLE 8.

Example 9 (3aR, 6aR) 2-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoicacid (3-oxo-hexahydrocyclopenta[b]furan-3a-yl)amide

HPLC Rt=23.67 mins (>80%), HPLC-MS 418.3 [M+H]⁺.

(a) Preparation of 2R-Bromo-4-methylpentanoic acid (Compound (17),Scheme 5)

A solution of sodium nitrite (5.1 g, 73 mmol) in water (15 ml) was addeddrop-wise at 0° C. over 5 hours to a stirred mixture of D-leucine (8.75g, 67 mmol), potassium bromide (29.75 g, 0.25 mol) and concentratedsulphuric acid (8.6 ml) in water (100 ml). The mixture was stirred for30 minutes at 0° C. then at ambient temperature for 20 hours. Theproduct was extracted into diethyl ether (2×150 ml) then the combinedethereal layers were washed with saturated aqueous sodium chloridesolution (2×100 ml), dried (MgSO₄) and the solvent removed in vacuo. Theresidue was purified by flash chromatography over silica gel elutingwith a gradient of methanol:dichloromethane 1:50→1:20. Appropriatefractions were combined and the solvents removed in vacuo to leave2R-bromo-4-methylpentanoic acid (17) as a colourless oil, yield 1.60 g,(12.3%). TLC (single spot, Rf=0.2, methanol:dichloromethane 1:20).Additionally, a second crop (5.2 g, 40%) of slightly impure product wasobtained

δ_(H) (400 MHz, CDCl₃ at 298K), 0.95 and 0.99 (both 3H, CH ₃CH, d,J=6.55 Hz), 1.77-1.89 (1H, CH₃CH, m), 1.93 (2Hβ, m), 4.31 (1Hα, t, J=7.7Hz), 9.3 (1H, CO₂ H, brs).

(b) Preparation of 2S-(4-tert-butylbenzylsulfanyl)-4-methylpentanoicacid (Compound (19), Scheme 5)

A solution of 2R-bromo-4-methylpentanoic acid (compound (17), 1.1 g, 5.6mmol) and (4-(tert-butyl)phenyl)methanethiol (1.0 g, 5.6 mmol) indimethylformamide (15 ml) was purged with nitrogen for 5 minutes thencooled to 0° C. Triethylamine (0.79 ml, 5.7 mmol) was added drop-wiseover 1 minute then the mixture was stirred for two days at ambienttemperature. The solvents were removed in vacuo and residue purified byflash chromatography over silica gel eluting with a gradient ofmethanol:dichloromethane 0:1→1:20. Appropriate fractions were combinedand the solvents removed in vacuo to leave a residue which was purifiedby flash chromatography over silica gel eluting with ethylacetate:heptane 2:5. Appropriate fractions were combined and thesolvents removed in vacuo to give2S-(4-tert-butylbenzylsulfanyl)-4-methylpentanoic acid (19) as acolourless oil, yield 150 mg, (9%). TLC (single spot, Rf=0.2,heptane:ethyl acetate 5:2), analytical HPLC with main peak Rt=22.117mins, HPLC-MS (main UV peak with Rt=11.072 mins, 317.2 [M+Na]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K), 0.70 and 0.85 (both 3H, CH ₃CH, d,J=6.3), 1.29 (9H, (CH ₃)₃C, s), 1.44-1.51 (1H, CH₃CH, m), 1.62-1.75(2Hβ, m), 3.15-3.20 (1Hα, m), 3.81 and 3.88 (both 1H, SCH ₂, d, J=13.2Hz), 7.25-7.35 (4H, aromatic).

Compound (19) was coupled under standard conditions to loaded buildingblock-linker construct (11) (following standard removal of Fmoc), thencleaved to provide EXAMPLE 9.

Example 10 (3aR, 6aR)2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-amide

HPLC Rt=21.86 mins (>80%), HPLC-MS 450.2 [M+H]⁺.

Scheme 9. Compound (19) was coupled under standard conditions to loadedbuilding block-linker construct (11). (following standard removal ofFmoc). The intermediate loaded thioether (1.2 μmole gear) was oxidisedwith m-chloroperbenzoic acid (5 eq, 65% reagent, 1.6 mg) indichloromethane (200 μL) for 5 hrs, followed by standard washing andthen cleaved to provide EXAMPLE 10.

Example 11 (3aR, 6aR)2-Cyclohexylethyl-4-morpholinyl-4-oxo-N-(3-oxo-hexahydro-cyclopenta[b]furan-3a-yl)-butyramide

HPLC-MS 407.1 [M+H]⁺, 835.1 [2M+Na]⁺

(a) Preparation of 2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyricacid methyl ester.

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.HCl (1.12 g, 5.69 mmol)then 1-hydroxybenzotriazole monohydrate (0.87 g, 5.69 mmol) were addedto a stirred solution of 2R-(cyclohexylmethyl)succinic acid 1-methylester (compound (24), 1.0 g, 4.38 mmol) in dimethylformamide (10 ml) at0° C. under argon. The mixture was stirred for 25 minutes thenmorpholine (0.7 ml, 8.76 mmol) was added drop-wise over 1 minute andstirring continued at ambient temperature for 16 hours. The product wasextracted into ethyl acetate (200 ml) then washed with 1.0M hydrochloricacid (3×100 ml), saturated aqueous sodium hydrogen carbonate solution(3×100 ml), water (100 ml), then saturated aqueous sodium chloridesolution (100 ml), dried (MgSO₄), and the solvent removed in vacuo togive 2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid methylester as an off-white solid, yield 1.22 g, (94%). HPLC-MS (single peakwith Rt=7.91 mins, 298.1 [M+H]⁺, 617.3 [2M+Na]⁺).

(b) Preparation of 2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyricacid (Compound (25), Scheme 7).

A solution of lithium hydroxide monohydrate (0.51 g, 12.18 mmol) inwater (27 ml) was added a stirred solution of2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid methyl ester(1.21 g, 4.06 mmol) in tetrahydrofuran (55 ml) and methanol (27 ml) at0° C. The mixture was stirred at ambient temperature for 1 hours thendiluted with water (100 ml). The aqueous layer was extracted withdiethyl ether (2×50 ml) which was discarded, then acidified to pH=1-2with 1M hydrochloric acid. The product was extracted intodichloromethane (3×50 ml), then the combined ethereal layers washed withwater (2×50 ml), saturated aqueous sodium chloride solution (2×50 ml),dried (MgSO₄) and the solvent removed in vacuo to leave a residue. Theresidue was purified by chromatography over silica gel eluting with agradient of methanol:dichloromethane 1:100→3:100. Appropriate fractionswere combined and the solvents removed in vacuo was to give2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid (25) as a whitesolid, yield 0.82 g, (71%). HPLC-MS (single peak with Rt=6.769 mins,284.2 [M+H]⁺, 589.2 [2M+Na]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K), 0.77-0.90 (2H, CH ₂(cyclohexane), m),1.05-1.40 (4H, CH ₂(cyclohexane), m), 1.50-1.90 (7H, CH(cyclohexane),CH(cyclohexane), m), 2.30-2.44 (2Hβ, m), 2.64-2.77 (1Hα, m), 2.96-3.10(1H, OH, brs), 3.40-3.78 (8H, CH ₂OCH ₂ and CH ₂NCH ₂, m).

Compound (25) was coupled under standard conditions to loaded buildingblock-linker construct (11) (following standard removal of Fmoc), thencleaved to provide EXAMPLE 11.

Example 12 (3aR, 6aR) 2-Biphenyl-3-yl-4-methyl-pentanoic acid(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)-amide

HPLC Rt=20.53 mins (>90%), HPLC-MS 392.3 [M+H]⁺

(a) Preparation of Biphenyl-3-yl-acetic acid methyl ester (Compound(27), Scheme 8)

Concentrated sulphuric acid (588 μL) was added to a solution of3-bromophenyl acetic acid (10 g, 46.5 mmol) in methanol (100 mL). Themixture was refluxed for 1.5 h and then cooled to ambient temperatureand evaporated under reduced pressure to afford a residue. The residuewas redissolved in diethyl ether (500 mL), washed with water (2×100 mL),brine (100 mL), dried (MgSO₄) and then evaporated under reduced pressureto afford 3-bromophenyl acetic acid methyl ester (10.65 g). The3-bromophenyl acetic acid methyl ester was dissolved in toluene (117 mL)then phenyl boronic acid (6.8 g, 55.69 mmol) added, followed by aaqueous solution of sodium carbonate (93 mL, 2M) andtetrakis(triphenylphosphine)palladium (1.6 g, 1.41 mmol). The mixturewas stirred overnight then cooled to ambient temperature and an aqueoussolution of saturated ammonium chloride (100 mL) added. The mixture wasextracted with ethyl acetate (2×200 mL), died (Na₂SO₄) and evaporatedunder reduced pressure to afford a residue. Flash chromatography of theresidue over silica (200 g) using ethyl acetate:heptane (3:48) as theeluent gave biphenyl-3-yl acetic acid methyl ester, yield 10.5 g, (99%),TLC (single UV spot, R_(f)=0.24, 10% ethyl acetate in heptane),analytical HPLC R_(t)=19.55 min, HPLC-MS (single main UV peak withR_(t)=9.35 min, 227.1 [M+H]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K) 3.76 (2H, s, CH₂CO₂CH₃), 3.77 (3H, s,OCH₃), 7.34-7.66 (9H, m, biphenyl-3-yl).

(b) Preparation of Biphenyl-3-yl-acetic acid

Water (39 mL), followed by lithium hydroxide monohydrate (4.2 g, 101.5mmol) were added to a solution of biphenyl-3-yl acetic acid methyl ester(11.43 g, 50.57 mmol) in methanol (265 mL). The mixture was stirred atambient temperature for 2 h then the organics were removed under reducedpressure. The mixture was acidified with dilute hydrochloric acid (1M,80 mL), extracted with chloroform (2×100 mL), dried (MgSO4) andevaporated under reduced pressure to afford biphenyl-3-yl acetic acid asa white solid, yield 10.6 g, (99%), analytical HPLC R_(t)=16.565 min,HPLC-MS (single main UV peak with R_(t)=7.91 min, 213.1 [M+H]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K) 3.77 (2H, s, CH₂CO₂CH₃), 7.28-7.52 (9H,m, biphenyl-3-yl).

(c) Preparation of 2RS-Biphenyl-3-yl-4-methylpent-4-enoic acid

A solution of biphenyl-3-yl acetic acid (7.0 g, 33 mmol) in anhydroustetrahydrofuran (84 mL) was added dropwise to a solution of lithiumdiisopropyl amide (36.4 mL, 2M solution in hexanes) in anhydroustetrahydrofuran (84 mL) at −78° C. The mixture was allowed to warm to 0°C. and stirred for 40 min. The mixture was then cooled to −78° C. and3-bromo-2-methylpropene (4.97 mL) rapidly added. The mixture was stirredfor 1 h at −78° C. then water (28 mL) added and the organics removedunder reduced pressure. The mixture was then acidified with hydrochloricacid (6M, 14 ml), extracted with ethyl acetate (3×100 ml), dried (MgSO4)and evaporated under reduced pressure to afford a residue. Flashchromatography of the residue over silica (400 g) usingmethanol:dichloromethane (3:97) as the eluent afforded impure2-biphenyl-3-yl-4-methylpentenoic acid (8.3 g). Flash chromatographyover silica (400 g) using methanol:dichloromethane (1.5:98.5) affordedpure 2-biphenyl-3-yl-4-methylpent-4-enoic acid, yield 5.27 g, (60%), TLC(single UV spot, R_(f)=0.28, 5% methanol in dichloromethane), analyticalHPLC R_(t)=19.99 min, HPLC-MS (single main UV peak with R_(t)=9.57 min,267.1 [M+H]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K), 1.765 (3H, s, CH₃), 2.53 (1H, dd, J=6.6and 14.7 Hz, 3-H₁), 2.91 (1H, dd, J=8.9 and 14.7 Hz, 3-H₁), 3.92 (1H,dd, J=6.6 and 8.9 Hz, 2-H), 4.79 (2H, d, J=10.7 Hz, 5-H₂), 7.30-7.62(9H, m, biphenyl-3-yl).

(d) Preparation of 2RS-Biphenyl-3-yl-4-methylpentanoic acid (Compound(28), Scheme 8)

Palladium on carbon (10%, 300 mg) was added portionwise to a solution of2RS-biphenyl-3-yl-4-methylpent-4-enoic acid (1 g, 3.76 mmol) in ethanol(40 mL) at 0° C. A hydrogen atmosphere was then introduced and themixture allowed to warm to ambient temperature. The mixture was stirredfor 18 h, then the hydrogen atmosphere removed and the mixture filteredover Celite and the catalyst washed with ethanol (40 mL). The combinedorganic filtrate was concentrated under reduced pressure to afford aresidue, which was flash chromatographed over silica (150 g) usingmethanol:dichloromethane (1:99) as the eluent to afford2RS-biphenyl-3-yl-4-methylpentanoic acid, yield 980 mg, (98%), TLC(single UV spot, R_(f)=0.45, 5% methanol in dichloromethane), analyticalHPLC R_(t)=20.92 min, HPLC-MS (single main UV peak with R_(t)=10.15 min,269.1 [M+H]⁺, 291.1 [M+Na]⁺).

δ_(H) (400 MHz, CDCl₃ at 298K), 0.93 (6H, d, J=6.6 Hz, 2×CH₃), 1.52-1.57(1H, m, 4-H₁), 1.71-1.76 (1H, m, 3-H₁), 1.97-2.05 (1H, m, 3-H₁), 3.66(1H, t, J=7.8 Hz, 2-H₁), 7.32-7.60 (9H, m, biphenyl-3-yl).

Compound (28) was coupled under standard conditions to loaded buildingblock-linker construct (11), then cleaved to provide EXAMPLE 12.

Example A Assays for Cysteine Protease Activity

The compounds of this invention may be tested in one of a number ofliterature based biochemical assays that are designed to elucidate thecharacteristics of compound inhibition. The data from these types ofassays enables compound potency and the rates of reaction to be measuredand quantified. This information, either alone or in combination withother information, would allow the amount of compound required toproduce a given pharmacological effect to be determined.

General Materials and Methods

Unless otherwise stated, all general chemicals and biochemicals werepurchased from either the Sigma Chemical Company, Poole, Dorset, U.K. orfrom Fisher Scientific UK, Loughborough, Leicestershire, U.K. Absorbanceassays were carried out in flat-bottomed 96-well plates (Spectra;Greiner Bio-One Ltd., Stonehouse, Gloucestershire, U.K.) using aSpectraMax PLUS384 plate reader (Molecular Devices, Crawley, U.K.).Fluorescence high throughput assays were carried out in either 384-wellmicrotitre plates (Corning Costar 3705 plates, Fisher Scientific) or96-well ‘U’ bottomed Microfluor W1 microtitre plates (Thermo Labsystems,Ashford, Middlesex, U.K.). Fluorescence assays were monitored using aSpectraMax Gemini fluorescence plate reader (Molecular Devices). Forsubstrates employing either a 7-amino-4-methylcoumarin (AMC) or a7-amino-4-trifluoromethylcoumarin (AFC) fluorophore, assays weremonitored at an excitation wavelength of 365 nm and an emissionwavelength of 450 nm and the fluorescence plate reader calibrated withAMC. For substrates employing a 3-amino-benzoyl (Abz) fluorophore,assays were monitored at an excitation wavelength of 310 nm and anemission wavelength of 445 mm; the fluorescence plate reader calibratedwith 3-amino-benzamide (Fluka). Unless otherwise indicated, all thepeptidase substrates were purchased from Bachem UK, St. Helens,Merseyside, UK. Substrates utilizing fluorescence resonance energytransfer methodology (i.e. FRET-based substrates) were synthesized atIncenta Limited using published methods (Atherton & Sheppard, SolidPhase Peptide Synthesis, IRL Press, Oxford, U.K., 1989) and employed Abz(2-aminobenzoyl) as the fluorescence donor and 3-nitro-tyrosine[Tyr(NO₂)] as the fluorescence quencher (Meldal, M. and Breddam, K.,Anal. Biochem., 195, 141-147, 1991). Hydroxyethylpiperazineethanesulfonate (HEPES), tris-hydroxylmethyl aminomethane (tris) base,bis-tris-propane and all the biological detergents (e.g. CHAPS,zwittergents, etc.) were purchased from CN Biosciences UK, Beeston,Nottinghamshire, U.K. Glycerol was purchased from Amersham PharmaciaBiotech, Little Chalfont, Buckinghamshire, U.K. Stock solutions ofsubstrate or inhibitor were made up to 10 mM in 100% dimethylsulfoxide(DMSO) (Rathburns, Glasgow, U.K.) and diluted as appropriately required.In all cases the DMSO concentration in the assays was maintained at lessthan 1% (vol./vol.).

Assay protocols were based on literature precedent (Table1; Barrett, A.J., Rawlings, N. D. and Woessner, J. F., 1998, Handbook of ProteolyticEnzymes, Academic Press, London and references therein) and modified asrequired to suit local assay protocols. Enzyme was added as required toinitiate the reaction and the activity, as judged by the change influorescence upon conversion of substrate to product, was monitored overtime. All assays were carried out at 25±1° C.

TABLE 1 The enzyme assays described herein were carried out according toliterature precedents. Enzyme Buffer Substrate Reference Cathepsin B IZ-Phe-Arg-AMC a, b Cathepsin H II Bz-Phe-Val-Arg-AMC a, b Cathepsin L IAc-Phe-Arg-AMC b, c Cathepsin S I Boc-Val-Leu-Lys-AMC c, d Caspase 1 IIIAc-Leu-Glu-His-Asp-AMC e Caspase 2 III Z-Val-Asp-Val-Ala-Asp-AFC fCaspase 3 III Ac-Asp-Glu-Val-Asp-AMC g, h Caspase 4 IIISuc-Tyr-Val-Ala-Asp-AMC f Caspase 5 III Ac-Leu-Glu-His-Asp-AMC Caspase 6III Ac-Val-Glu-Ile-Asp-AMC i, j, k Caspase 7 III Ac-Asp-Glu-Val-Asp-AMCCaspase 8 III Ac-Ile-Glu-Thr-Asp-AMC l Caspase 9 IIIAc-Leu-Glu-His-Asp-AMC Caspase 10 III Ac-Ile-Glu-Thr-Asp-AMC CruzipainIV D-Val-Leu-Lys-AMC m, n CPB2.8ΔCTE XI Pro-Phe-Arg-AMC q S. Aureus IAbz-Ile-Ala-Ala-Pro- o Extracellular Tyr(NO₂)-Glu-NH₂ cysteine peptidaseClostripain Z-Gly-Gly-Arg-AMC p FMDV LP V Abz-Arg-Lys-Leu-Lys-Gly- rAla-Gly-Ser-Tyr(NO₂)-Glu- NH₂ Trypsin VI Z-Gly-Gly-Arg-AMC s Calpain μVII Abz-Ala-Asn-Leu-Gly-Arg-Pro- t Ala-Leu-Tyr(NO₂)-Asp-NH₂ Calpain mVIII Abz-Lys-Leu-Cys(Bzl)-Phe-Ser- t Lys-Gln-Tyr(NO₂)-Asp-NH₂ CathepsinK IX Z-Phe-Arg-AMC u Cathepsin X X v, w I 10 mM BTP, pH 6.5 containing 1mM EDTA, 5 mM 2-mercaptoethanol and 1 mM CaCl₂ II 10 mM BTP, pH 6.5containing 1 mM EDTA, 142 mM NaCl, 1 mM DTT, 1 mM CaCl₂, 0.035 mMZwittergent 3-16 III 50 mM HEPES pH 7.2, 10% Glycerol, 0.1% CHAPS, 142mM NaCl, 1 mM EDTA, 5 mM DTT IV 100 mM sodium phosphate, pH 6.75containing 1 mM EDTA and 10 mM L-cysteine V 50 mM trisacetate, pH 8.4containing 1 mM EDTA, 10 mM L-cysteine and 0.25% (w/v) CHAPS VI 10 mMHEPES, pH 8.0 containing 5 mM CaCl₂ VII 10 mM HEPES, pH 7.5 containing 2mM 2-mercaptoethanol and 100 μM CaCl₂ VIII 10 mM HEPES, pH 7.5containing 2 mM 2-mercaptoethanol and 200 μM CaCl₂ IX 100 mM sodiumacetate; pH 5.5 containing 10 mM L-cysteine and 1 mM EDTA X 100 mMsodium acetate; pH 5.5 containing 10 mM L-cysteine; 0.05% (w/v) Brij 35and 1 mM EDTA XI 100 mM sodium acetate; pH 5.5 containing 10 mML-cysteine; 142 mM sodium chloride and 1 mM EDTA a Barrett, A. J.,Biochem. J., 187, 909-912, 1980 b Barrett, A. J. and Kirschke, H.,Methods Enzymol., 80, 535-561, 1981 c Quibell, M. and Taylor, S.,WO0069855, 2000 d Bromme, D., Steinert, ., Freibe, S., Fittkau, S.,Wiederanders, B., and Kirschke, H., Biochem. J., 264, 475-481, 1989 eRano, T. A., et. al., Chem. Biol., 4, 149, 1997 f Talanian, R. V., et.al., J. Biol. Chem., 272, 9677, 1997 g Lazebnik, Y. A., Kaufmann, S. H.,Desnoyers, S., Poirer, G. G. and Earnshaw, W. C., Nature, 371, 768-774,1994 h Han, Z., et. al., J. Biol. Chem., 272, 13432, 1997 i Takahashi,A., et. al., PNAS, 93, 8395, 1996 j Martins, L. M., et. al., J. Biol.Chem., 272, 7421, 1997 k Nagata, S., Cell., 88, 355, 1997 l Harris, J.L., et. al., J. Biol. Chem., 273, 27364, 1998 m Cazzulo, J. J., CazzuloFranke, M. C., Martinez, J. and Franke de Cazzulo, B. M., Biochim.Biophys. Acta., 1037, 186-191, 1990 n Cazzulo, J. J., Bravo, M.,Raimondi, A., Engstrom, U., Lindeberg, G. and Hellman, U., Cell Mol.Biol., 42, 691-696, 1996 o Potempa, J., Dubin, A., Korzus, G. andTravis, J., Biochem. J., 263, 2664-2667, 1988 p Kembhavi, A. A., Buttle,D. J., Rauber, P. and Barrett, A. J., FEBS Lett., 283, 277-280, 1991 qAlves, L. C., et. al., Mol. Biochem. Parasitol, 116, 1-9, 2001. rGuarné, et. al., J. Mol. Biol., 302, 1227-1240, 2000. s Halfon andCraik, (Barret, Rawlings and Woessner, eds.), in Handbook of ProteolyticEnzymes, Academic Press, London, 12-21, 1998. t Sasaki, et. al., (1984),J. Biol. Chem., 259, 12489-12494, 1984. u Bossard, M. J., et. al., , J.Biol. Chem., 21, 12517-12524, 1996 v Santamaria, I., et. al., J. Biol.Chem., 273, 16816-16823, 1998 w Klemencic, J, et al., Eur. J. Biochem.,267, 5404-5412, 2000Trypanosoma cruzi Cruzipain Peptidase Activity Assays

Wild-type cruzipain, derived from Trypanosoma cruzi Dm28 epimastigotes,was obtained from Dr. Julio Scharfstein (Instituto de Biofisica CarlosChagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro,Brazil). Activity assays were carried out in 100 mM sodium phosphate, pH6.75 containing 1 mM EDTA and 10 mM L-cysteine using 2.5 mM enzyme.Ac-Phe-Arg-AMC (K_(M) ^(app)≈12 μM) and D-Val-Leu-Lys-AMC (K_(M)^(app)≈4 μM) were used as the substrates. Routinely, Ac-FR-AMC was usedat a concentration equivalent to K_(M) ^(app) and D-Val-Leu-Lys-AMC wasused at a concentration of 25 μM. The rate of conversion of substrate toproduct was derived from the slope of the increase in fluorescencemonitored continuously over time.

Leishmania mexicana Cysteine Protease B (CPB) Peptidase Activity Assays

Wild-type recombinant CPB without the C-terminal extention (i.e.CPB2.8ΔCTE; Sanderson, S. J., et. al., Biochem J., 347, 383-388, 2000)was obtained from Dr. Jeremy Mottram (Wellcome Centre for MolecularParasitology, The Anderson College, University of Glasgow, Glasgow,U.K.). Activity assays were carried out in 100 mM sodium acetate; pH 5.5containing 1 mM EDTA; 200 mM NaCl and 10 mM DTT (Alves, L. C., et. al.,Mol. Biochem. Parasitol, 116, 1-9, 2001) using 0.25 nM enzyme.Pro-Phe-Arg-AMC (K_(M) ^(app)≈38 μM) was used as the substrate at aconcentration equivalent to K_(M) ^(app). The rate of conversion ofsubstrate to product was derived from the slope of the increase influorescence monitored continuously over time.

Cathepsin Peptidase Activity Assays

Bovine cathepsin S, human cathepsin L, human cathepsin H and humancathepsin B were obtained from CN Biosciences. Recombinant humancathepsin S, human cathepsin K and human cathepsin X were obtained fromDr. Boris Turk (Josef Stefan Institute, Ljubljana, Slovenia). Unlessotherwise stated, all peptidase activity assays were carried out in 10mM bis-tris-propane (BTP), pH 6.5 containing 1 mM BDTA, 5 mM2-mercaptoethanol and 1 mM CaCl₂. Human cathepsin H activity assays werecarried out in 10 mM BTP pH 6.5, 142 mM NaCl₂, 1 mM CaCl₂, 1 mM EDTA, 1mM DTT, 0.035 mM Zwittergent 3-16. Human cathepsin K assays were carriedout in 100 mM sodium acetate; pH 5.5 containing 20 mM L-cysteine and 1mM EDTA (Bossard, M. J., et. al., J. Biol. Chem., 21, 12517-12524,1996). Human cathepsin X assays were carried out in 100 mM sodiumacetate; pH 5.5 containing 20 mM L-cysteine; 0.05% (w/v) Brij 35 and 1mM EDTA (Santamaria, I., et. al., J. Biol. Chem., 273, 16816-16823,1998; Klemencic, J, et al., Eur. J. Biochem., 267, 5404-5412, 2000). Thefinal enzyme concentrations used in the assays were 0.5 nM bovinecathepsin S, 1 nM cathepsin L, 0.1 nM cathepsin B, 0.25 nM Cathepsin K;1 nM cathepsin X and 10 nM cathepsin H. For the inhibition assays, thesubstrates used for cathepsin S, cathepsin L, cathepsin B, cathepsin Kand cathepsin H were boc-Val-Leu-Lys-AMC (K_(M) ^(app)≈30 μM),Ac-Phe-Arg-AMC (K_(M) ^(app)≈20 μM), Z-Phe-Arg-AMC (K_(M) ^(app)≈40 μM),Z-Leu-Arg-AMC (K_(M) ^(app)≈2 μM); Bz-Phe-Val-Arg-AMC (K_(M) ^(app)≈150μM) respectively. In each case the substrate concentration used in eachassay was equivalent to the K_(M) ^(app). The rate of conversion ofsubstrate to product was derived from the slope of the increase influorescence monitored continuously over time.

Trypsin Peptidase Activity Assays

Human pancreatic trypsin (iodination grade; CN Biosciences) activityassays were carried out in 10 mM HEPES, pH 8.0 containing 5 mM CaCl₂using 0.1 nM trypsin. For the inhibition assays, Z-Gly-Gly-Arg-AMC(K_(M) ^(app)≈84 μM) was used as the substrate at a concentrationequivalent to K_(M) ^(app). The rate of conversion of substrate toproduct was derived from the slope of the increase in fluorescencemonitored continuously over time.

Clostripain Peptidase Activity Assays

Clostripain (Sigma) activity assays were carried out in 10 mM BTP, pH6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol and 1 mM CaCl₂ using0.3 nM enzyme. For the inhibition assays, Z-Gly-Gly-Arg-AMC (K_(M)^(app)≈100 μM) was used as the substrate at a concentration equivalentto K_(M) ^(app). The rate of conversion of substrate to product wasderived from the slope of the increase in fluorescence monitoredcontinuously over time.

Calpain Peptidase Activity Assays

Calpain (human erythrocyte μ-calpain and porcine kidney m-calpain; CNBiosciences) activity assays were carried out in 10 mM HEPES, pH 7.5containing 2 mM 2-mercaptoethanol and CaCl₂ using 25 nM of either enzyme(Sasaki, et. al., J. Biol. Chem., 259, 12489-12494, 1984). For μ-calpaininhibition assays, the buffer contained 100 μM CaCl₂ andAbz-Ala-Asn-Leu-Gly-Arg-Pro-Ala-Leu-Tyr(NO₂)-Asp-NH₂ (K_(M) ^(app)≈20μM; Incenta Limited) was used as the substrate. For m-calpain inhibitionassays, the assay buffer contained 200 μM CaCl₂ andAbz-Lys-Leu-Cys(Bzl)-Phe-Ser-Lys-Gln-Tyr(NO₂)-Asp-NH₂ (K_(M) ^(app)≈22μM; Incenta Limited) was used as the substrate. In both cases thesubstrate concentration employed in the assays was equivalent to theK_(M) ^(app). The rate of conversion of substrate to product was derivedfrom the slope of the increase in fluorescence monitored continuouslyover time.

Extracellular S. aureus V8 Cysteine Peptidase (Staphylopain) PeptidaseActivity Assays

S. aureus V8 was obtained from Prof. S. Arvidson, Karolinska Institute,Stockholm, Sweden. Extracellular S. aureus V8 cysteine peptidase(staphylopain) activity assays were carried out using partially purifiedS. aureus V8 culture supernatant (obtained from Dr. Peter Lambert, AstonUniversity, Birmingham, U.K.). Activity assays were carried out in 10 mMBTP, pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol and 1 mM CaCl₂using two-times diluted partially purified extract. For the inhibitionassays, Abz-Ile-Ala-Ala-Pro-Tyr(NO₂)-Glu-NH₂ (K_(M) ^(app)≈117 μM;Incenta Limited) was used as the substrate at a concentration equivalentto K_(M) ^(app). The rate of conversion of substrate to product wasderived from the slope of the increase in fluorescence monitoredcontinuously over time.

Foot-and-mouth Disease Leader Peptidase (FMDV-LP) Activity Assays

Recombinant wild-type FMDV-LP was obtained from Dr. Tim Skern (Institutfür Medizinische Biochemie, Abteilung für Biochemie, Universtät Wien,Wien, Austria). Activity assays were carried out in 50 mM trisacetate,pH 8.4 containing 1 mM EDTA, 10 mM L-cysteine and 0.25% (w/v) CHAPSusing 10 nM enzyme. For the inhibition assays,Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser-Tyr(NO₂)-Glu-NH₂ (K_(M) ^(app)≈51μM, Incenta Limited) was used as the substrate at a concentrationequivalent to K_(M) ^(app). The rate of conversion of substrate toproduct was derived from the slope of the increase in fluorescencemonitored continuously over time.

Caspase Peptidase Activity Assays

Caspases 1-10 were obtained from CN Biosciences or BioVision Inc.(Mountain View, Calif., USA) and all assays were carried out in 50 mMHEPES; pH 7.2, 10% (v/v) glycerol, 0.1% (w/v) CHAPS, 142 mM NaCl, 1 mMEDTA, 5 mM dithiothreitol (DTT) using 0.1-1 U per assay. For caspase 1,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 2,Z-Val-Asp-Val-Ala-Asp-AFC was used as the substrate; for caspase 3,Ac-Asp-Glu-Val-Asp-AMC was used as the substrate; for caspase 4,Suc-Tyr-Val-Ala-Asp-AMC was used as the substrate; for caspase 5,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 6,Ac-Val-Glu-Ile-Asp-AMC was used as the substrate; for caspase 7,Ac-Asp-Glu-Val-Asp-AMC was used as the substrate; for caspase 8,Ac-Ile-Glu-Thr-Asp-AMC was used as the substrate; for caspase 9,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 10,Ac-Ile-Glu-Thr-Asp-AMC was used as the substrate (Nicholson, D. W. andThomberry, N. A., TIBS, 22, 299-306, 1997; Stennicke, H. R. andSalvesen, G. S., J. Biol. Chem., 272(41), 25719-25723, 1997; Talanian,R. V., et. al., J. Biol. Chem., 272(15), 9677-9682, 1997; Wolf, B. B.and Green, D. R., J. Biol. Chem., 274(29), 20049-20052, 1999). The rateof conversion of substrate to product was derived from the slope of theincrease in fluorescence monitored continuously over time.

Measurement of the Apparent Macroscopic Binding (Michaelis) Constants(K_(M) ^(app)) for Substrates

The apparent macroscopic binding constant (K_(M) ^(app)) for eachsubstrate was calculated, from the dependence of enzyme activity as afunction of substrate concentration. The observed rates were plotted onthe ordinate against the related substrate concentration on the abscissaand the data fitted by direct regression analysis (Prism v 3.02;GraphPad, San Diego, USA) using Equation 1 (Cornish-Bowden, A.Fundamentals of enzyme kinetics Portland Press; 1995, 93-128.).$\begin{matrix}{v_{i} = \frac{V_{\max}^{app} \cdot \left\lbrack S_{o} \right\rbrack}{\left\lbrack S_{o} \right\rbrack + K_{M}^{app}}} & (1)\end{matrix}$

In Equation 1 ‘v_(i)’ is the observed initial rate, ‘V_(max) ^(app)’ isthe observed maximum activity at saturating substrate concentration,‘K_(M) ^(app)’ is the apparent macroscopic binding (Michaelis) constantfor the substrate, ‘[S_(o)]’ is the initial substrate concentration.

Measurement of the Inhibition Constants

The apparent inhibition constant (K_(i)) for each compound wasdetermined on the basis that inhibition was reversible and occurred by apure-competitive mechanism. The K_(i) values were calculated, from thedependence of enzyme activity as a function of inhibitor concentration,by direct regression analysis (Prism v 3.02) using Equation 2(Cornish-Bowden, A., 1995.). $\begin{matrix}{v_{i} = \frac{V_{\max}^{app} \cdot \lbrack S\rbrack}{\lbrack S\rbrack + \left\{ {K_{M}^{app} \cdot \left( {\lbrack I\rbrack/K_{i}} \right)} \right\}}} & (2)\end{matrix}$

In Equation 2 ‘v_(i)’ is the observed residual activity, ‘V_(max)^(app)’ is the observed maximum activity (i.e. in the absence ofinhibitor), ‘K_(M) ^(app)’ is the apparent macroscopic binding(Michaelis) constant for the substrate, ‘[S]’ is the initial substrateconcentration, ‘K_(i)’ is the apparent dissociation constant and ‘[I]’is the inhibitor concentration.

In situations where the apparent dissociation constant (K_(i) ^(app))approached the enzyme concentrations, the K_(i) ^(app) values werecalculated using a quadratic solution in the form described by Equation3 (Morrison, J. F. Trends Biochem. Sci., 7, 102-105, 1982; Morrison, J.F. Biochim. Biophys. Acta, 185, 269-286, 1969; Stone, S. R. andHofsteenge, J. Biochemistry, 25, 4622-4628, 1986). $\begin{matrix}{v_{i} = \frac{F\left\{ {E_{o} - I_{o} - K_{i}^{app} + \sqrt{\left( {E_{o} - I_{o} - K_{i}^{app}} \right)^{2} + {4 \cdot K_{i}^{app} \cdot E_{o}}}} \right\}}{2}} & (3)\end{matrix}$  K _(i) ^(app) =K _(i)(1+[S _(o) ]/K _(M) ^(app))  (4)

In Equation 3 ‘v_(i)’ is the observed residual activity, ‘F’ is thedifference between the maximum activity (i.e. in the absence ofinhibitor) and minimum enzyme activity, ‘E_(o)’ is the total enzymeconcentration, ‘K_(i) ^(app)’ is the apparent dissociation constant and‘I_(o)’ is the inhibitor concentration. Curves were fitted by non-linearregression analysis (Prism) using a fixed value for the enzymeconcentration. Equation 4 was used to account for the substratekinetics, where ‘K_(i)’ is the inhibition constant, ‘[S_(o)]’ is theinitial substrate concentration and ‘K_(M) ^(app)’ is the apparentmacroscopic binding (Michaelis) constant for the substrate (Morrison,1982).

The Second-order Rate of Reaction of Inhibitor with Enzyme

Where applicable, the concentration dependence of the observed rate ofreaction (k_(obs)) of each compound with enzyme was analysed bydetermining the rate of enzyme inactivation under pseudo-first orderconditions in the presence of substrate (Morrison, J. F., TIBS, 102-105,1982; Tian, W. X. and Tsou, C. L., Biochemistry, 21, 1028-1032, 1982;Morrison, J. F. and Walsh, C. T., from Meister (Ed.), Advances inEnzymol., 61, 201-301, 1988; Tsou, C. L., from Meister (Ed.), Advancesin Enzymol., 61, 381-436, 1988;). Assays were carried out by addition ofvarious concentrations of inhibitor to assay buffer containingsubstrate. Assays were initiated by the addition of enzyme to thereaction mixture and the change in fluorescence monitored over time.During the course of the assay less than 10% of the substrate wasconsumed. $\begin{matrix}{F = {{v_{s}t} + \frac{\left( {v_{o} - v_{s}} \right)\left\lfloor {1 - {\mathbb{e}}^{({k_{obs} \cdot t})}} \right\rfloor}{k_{obs}} + D}} & (5)\end{matrix}$

The activity fluorescence progress curves were fitted by non-linearregression analysis (Prism) using Eq. 5 (Morrison, 1969; Morrison,1982); where ‘F’ is the fluorescence response, ‘t’ is time, ‘v_(o)’ isthe initial velocity, ‘v_(s)’ is the equilibrium steady-state velocity,‘k_(obs)’ is the observed pseudo first-order rate constant and ‘D’ isthe intercept at time zero (i.e. the ordinate displacement of thecurve). The second order rate constant was obtained from the slope ofthe line of a plot of k_(obs) versus the inhibitor concentration (i.e.k_(obs)/[I]). To correct for substrate kinetics, Eq. 6 was used, where‘[S_(o)]’ is the iniitial substrate concentration and ‘K_(M) ^(app)’ isthe apparent macroscopic binding (Michaelis) constant for the substrate.$\begin{matrix}{k_{inact} = \frac{k_{obs}\left( {1 + {\left\lbrack S_{o} \right\rbrack/K_{M}^{app}}} \right)}{\lbrack I\rbrack}} & (6)\end{matrix}$

Compounds of the invention were tested by the above described assays andobserved to exhibit cruzipain inhibitory activity or inhibitory activityagainst an alternative CA C1 cysteine protease with an in vitro Kiinhibitory constant of less than or equal to 100 μM. Exemplaryinhibition data for a number of example compounds of the invention aregiven in table 2.

TABLE 2 Exemplary inhibition data (Ki expressed as μM). Bovine HumanHuman EXAMPLE N ^(o) Cruzipain Cathepsin S Cathepsin L Cathepsin K 1<2 >50 >20 >100 7 >50 <2 >25 >50 6 >20 >25 >10 <2

1. A compound according to general formula (I):

wherein: R¹=C₀₋₇-alkyl (when C=0, R¹ is simply hydrogen),C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl (when C=0, R¹ is simply an aromaticmoiety Ar); Ar is an aromatic moiety which is a 5- or 6-memberedmonocyclic or 9- or 10-membered bicyclic ring, wherein the aromatic ringis optionally substituted; Z=O or S; Y=CR⁵R⁶—CO, where R⁵ and R⁶ areindependently chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl;(X)_(o)=CR⁷R⁸, where R⁷ and R⁸ are independently chosen from C₀₋₇-alkyl,C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and o is a number from zero to three;(W)_(n)=O, S, C(O), S(O) or S(O)₂ or NR⁹, where R⁹ is chosen fromC₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and n is zero or one;(V)_(m)=C(O), C(S), S(O), S(O)₂, S(O)₂NH, OC(O), NHC(O), NHS(O),NHS(O)₂, OC(O)NH, C(O)NH or CR¹⁰OR¹¹, where R¹⁰ and R¹¹ areindependently chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl andm is a number from zero to three, provided that when m is greater thanone, (V)_(m) contains a maximum of one carbonyl or sulphonyl group; U=astable 5- to 7-membered monocyclic or a stable 8- to 11-memberedbicyclic ring which is either saturated or unsaturated and whichincludes zero to four heteroatoms (as detailed below), said ringselected from:

wherein R¹² is: C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl,O—C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl, S—C₀₋₇-alkyl,S—C₃₋₆-cycloalkyl, S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-alkyl, NH—C₃₋₆-cycloalkyl,NH—Ar—C₀₋₇-alkyl, N(C₀₋₇-alkyl)₂, N(C₃₋₆-cycloalkyl)₂ orN(Ar—C₀₋₇-alkyl)₂ or, when it is part of the group CHR¹² or CR¹², R¹²may be halogen; A is chosen from: CH₂, CHR¹², O, S and NR¹³; wherein R¹²is as defined above and R¹³ is chosen from: C₀₋₇-alkyl, C₃₋₆-cycloalkyland Ar—C₀₋₇-alkyl; B, D and G are independently chosen from: CR¹², whereR¹² is as defined above, or N; E is chosen from: CH₂, CHR¹², O, S andNR¹³, where R¹² and R¹³ are defined as above; J, L, M, R, T, T₂, T₃ andT₄ are independently chosen from: CR¹² and N, where R¹² is as definedabove; T₅ is chosen from: CH or N; q is a number from one to three,thereby defining a 5-, 6- or 7-membered ring.
 2. A compound as claimedin claim 1 wherein Z is O.
 3. A compound as claimed in claim 1 whereinR¹ is C₀₋₇-alkyl or Ar—C₀₋₇-alkyl.
 4. A compound as claimed in claim 3wherein R¹ is selected from hydrogen or one of the following moieties:


5. A compound as claimed in claim 1 wherein Y is CR⁵R⁶CO where R⁵ and R⁶are independently selected from C₀₋₇-alkyl, C₃₋₆-cycloalkyl orAr—C₀₋₇-alkyl.
 6. A compound as claimed in claim 5 where Y is selectedfrom one of the following moieties:

wherein R¹², R¹³ and Ar are as defined above.
 7. A compound as claimedin claim 1 wherein Y is CHR⁶CO where R⁶ is Ar—CH₂—, where the aromaticring is an optionally substituted phenyl or monocyclic heterocycle.
 8. Acompound as claimed in claim 1 wherein Y is CHR⁶CO where R⁶ is abranched alkyl group or a straight heteroalkyl chain.
 9. A compound asclaimed in claim 1 wherein Y is CHR⁶CO where R⁶ is cyclohexylmethyl. 10.A compound as claimed in claim 1 wherein Y is selected from thefollowing:

wherein (X)_(o), R¹² and Ar are as defined previously.
 11. A compound asclaimed in claim 1 wherein, in the group (X)_(o), X is CR⁷R⁸ and each ofR⁷ and R⁸ is independently selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl.12. A compound as claimed in claim 1 wherein (X)_(o) is one of thefollowing moieties:

wherein R¹² and R¹³ are as defined previously.
 13. A compound as claimedin claim 1, wherein (X)_(o) is an alkyl group and where o=0 or
 1. 14. Acompound as claimed in claim 1 wherein, in the group (W)_(n): W is O, S,SO₂, SO, C(O) or NR⁹, where R⁹ is C₀₋₄-alkyl; and n is 0 or
 1. 15. Acompound as claimed in claim 1 wherein, in the group (W)_(n): W is O, S,SO₂, C(O) or NH, and n is 0 or
 1. 16. A compound as claimed in claim 1wherein, in the group (W)_(n): W is NH and n is
 1. 17. A compound asclaimed in claim 1 wherein, in the group (V)_(m): V is C(O), C(O)NH orCHR¹¹, where R¹¹ is C₀₋₄-alkyl; and m is 0 or
 1. 18. A compound asclaimed in claim 1 wherein the combination (V)_(m) and (W)_(m) is one ofthe following:


19. A compound as claimed in claim 18, wherein the combination (V)_(m)and (W)_(m) is one of the first eight structures depicted in claim 18.20. A compound as claimed in claim 18, wherein the combination (V)_(m)and (W)_(m) is the ninth structure depicted in claim
 18. 21. A compoundas claimed in claim 1 wherein the combination (X)_(o), (V)_(m) and(W)_(m) is one of the following:


22. A compound as claimed in claim 1 wherein U is an optionallysubstituted 5- or 6-membered saturated or unsaturated heterocycle or anoptionally substituted saturated or unsaturated 9- or 10-memberedheterocycle.
 23. A compound as claimed in claim 22 wherein U is one ofthe following:

wherein R¹² is as defined previously.
 24. A compound as claimed in claim1 wherein U is a bulky alkyl or aryl group at the para position of anaryl Ar.
 25. A compound as claimed in claim 1 wherein U is a meta orpara-biaryl Ar—Ar, where Ar is as previously defined.
 26. A compound asclaimed in claim 1, wherein U represents a group selected from:

where R¹², D, E, G, J, L, M, R, T, T₂, T₃ and T₄ are defined previously.27. A compound as claimed in claim 1, wherein U represents a groupselected from:

wherein R¹², D, E, G, J, L, M, R and T are as defined previously.
 28. Acompound as claimed in claim 1, wherein U represents a group selectedfrom:

wherein R¹², D, E, G, J and L are as defined previously.
 29. A method ofvalidating a known or putative cysteine protease as a therapeutictarget, the method comprising: (a) assessing the in vitro binding of acompound as claimed in claim 1 to an isolated known or putative cysteineprotease, providing a measure of ‘potency’; and optionally, one or moreof the steps of: (b) assessing the binding of a compound as claimed inclaim 1 to closely related homologous proteases of the target andgeneral house-keeping proteases (e.g. trypsin) to provides a measure of‘selectivity’; (c) monitoring a cell-based functional marker of aparticular cysteine protease activity, in the presence of a compound asclaimed in claim 1; and (d) monitoring an animal model-based functionalmarker of a particular cysteine protease activity, in the presence of acompound as claimed in claim
 1. 30. A composition comprising one or morecompounds as claimed in claim 1 and a pharmaceutically or veterinarilyacceptable carrier.
 31. A method for preventing or treating Chagas'disease comprising administering an effective amount of one or morecompounds of claim 1 to a patient in need of such prevention ortreatment.
 32. A compound as claimed in claim 8, wherein R⁶ is isobutyl.33. A compound as claimed in claim 8, wherein R⁶ is benzylsulfanylmethylor benzylsulphonylmethyl.
 34. A compound as claimed in claim 13, wherein(X)_(o) is methylene.
 35. A method for preventing or treating a diseaseresulting from elevated levels of cysteine protease selected from thegroup consisting of infections by Pneumocystis carinii, Trypsanomacruzi, Trypsanoma brucei, Crithidia fusiculata, Leishmania mexicana,Clostridium histolyticum and Staphylococcus aureas comprisingadministering to a patient in need of such prevention or treatment aneffective amount of one or more compounds as claimed in claim 1.